j^r?
CHARACTERIZATION OF THE FECAL INDICATOR
BACTERIAL FLORA OF SANITARY SEWAGE VVITI
APPLICATION TO IDENTIFYING THE PRESENCE 01
SANITARY WASTE IN STORM SEWERS
R. A. C. PROJECT NO. 247PL
H
Environment
Ontario
ISBN 0-7729-8769-6
CHARACTERIZATION OF THE FECAL INDICATOR
BACTERIAL FLORA OF SANITARY SEWAGE WITH
APPLICATION TO IDENTIFYING THE PRESENCE OF
SANITARY WASTE IN STORM SEWERS
R. A. C. PROJECT NO. 247PL
Prepared for Environment Ontario by:
Patricia Seyfried and Trudy Bleier
Department of Microbiology
University of Toronto
Elizabeth Harris
Lake Simcoe and Region Conversation Authority
Michael Young
Ministry of the Environment Laboratory Services
.SEPTEMBER 1991
o
RECYCLABLE
Cette publication technique n'est disponible qu'en anglais.
Copvright: Queen's printer for Ontario,
1991
This publication may be reproduced for
non-commercial purposes with appropriate
attnbution.
PIBS 1657
log 91-9300-274
ACKNOWLEDGEMENT AND DISCLAIMER
This report was prepared for the Ontario Ministry of the
Environment. The views and ideas expressed in this report
are those of the author and do not necessarily reflect
the views and policies of the Ministry of the
Environment, nor does mention of trade names or
commercial products constitute endorsement or
recommendation for use. The Ministry, however,
encourages the distribution of information and strongly
supports technology transfer and diffusion.
Any person who wishes to republish part or all of this
report should apply for permission to do so to the
Research and Technology Branch, Ontario Ministry of the
Environment, 135 St. Clair Avenue West, Toronto, Ontario,
M4V 1P5, Canada.
Copyright: Queen's Printer for Ontario
This publication may be reproduced for
non-commercial purposes with appropriate
attribution.
ACKNOWIEDGEMENT
Itds work was supported by research project nuinber 274 PL from the Ontario
Ministry of the Environment. The itanuscript was prepared by Elizabeth Harris,
Patricia Seyfried and Trudy Bleier. Technical assistance was provided by
Bhavna Madhvani, Anjini Prashad, Rita Harmandayan, Joanne Vavougios, Mona
Sidarous, Nella Mauceri, Julianna Giczi, Kay Auanjou, Lionel Noronha, Seema
Boodoosin^, Bruno Esposito, Kay Hoasjoe, Aisha SheiWi, Helen Qiao, In-ja Huh,
Jim Ng, Niva Kulendran, Eric Hani, Natalie Veitch, Eric Bauer, Arif Somani,
Frank Fassos, Yan Xu, Frank De Palma, Charles Wachsberg and Susan Davison.
Collection of samples by Mr. Alex Marich and employees of the City of
Toronto Public Works Department is gratefully acknowledged.
DISCIAIMER
This report has been prepared for the research Advisory Committee,
Ministry of the Environment in fulfillment of the terms of the grant. The
views ej^ressed herein are those of the authors and they do not necessarily
reflect the views and policies of the Ontario Ministry of the Environment.
HJBUCATIONS RELATED TO THIS roCJECT
Seyfried, P.L, E.M. Harris, I. Huh, R. Harmandayan and E. Hani 1987.
Characterization of the fecal indicator bacterial flora of sanitary sewage
with application to identifying the presence of sanitary waste in storm
sewers. Proceedings Technology Transfer Conference, Part D. Analytical
Methods. Royal York Hotel, Toronto, November 30, and December 1, 1987.
Sponsored by the Research Advisory Committee, Ministry of the Environment,
pp. 1-24.
- 11 -
Seyfried, P.L. , T. Bleier, Y. Xu and R. Harmandayan. 1988. C3Tarac±erization
of the fecal indicator bacterial flora of sanitary sewage with application
to identifying the presence of sanitary waste in storm sewers.
Proceedings Technology Transfer Conference, Session B, Water Quality
Research, Royal York Hotel, Toronto, November 28 and 29, 1988. Sponsored
by Research and Technology Branch, Environment Ontario, pp. 247-267.
- Ill -
ABsrnRAcr
storm sewers are designed to channel untreated storm water into surface
waters. An unusually hi<^ dry weather flew of storm sewage along with hi(^
fecal coliform counts irjiicate that there may be potentially hazardous sanitary
connections in the storm sewer line. Ihe c±ijective of this study was to assess
the use of bacterial indicators to trace illegal sanitary connections to storm
sewers. The indicators selected for study were Escherichia coli, fecal
coliforms, fecal streptococci, enterococci, Pseudomonas aeruginosa, Clostridium
perfrinqens, and Bifidobacterium sp. The organisms were collected during
periods of wet and dry weather from October, 1986 to August, 1988. The
sampling sites A, B and C in the Mount Steven Trunk storm sewer line were
selected because this area was designated a hi(^ priority sewer by the Ministry
of the Environment. Non-priority sites, labelled X, Y and Z in the Mount
Steven Trunk storm sewer branch lines were saitpled for conparison. For wet
weather, storm water run-off was collected at the X, Y and Z sites. Samples
were also obtained from a sanitary s^^er in close proximity to the priority
storm sewer sanpling points; these sites were labelled D, E and F.
Biochemical testing, serotyping, and/ or genotyping were used to further
characterize more than 4,000 fecal streptococcus, Pseudomonas aeruginosa , and
Bifidobacterium spp. isolates. Speciation of the fecal streptococci showed
that Streptococcus faecal is subsp. faecal is was more predominant in sanitary
and high priority sewers than in surface runoff and non-priority sewers.
Streptococcus faecium subsp. casseliflavus, on the other hand, was found
primarily in surface water runoff and non-priority storm sewers. Litmus milk
reactions among the S^ faecal is isolates generally did not assist in source
tracing. IXIA sequence studies of the fecal streptococci, using Restriction
- IV -
Endonuclease Analysis (REA) , produced many different restriction patterns and
it was difficult to establish any relationship between the isolates. Although
serotyping of Pseudomonas aeruginosa was found to produce nonspecific results,
genotyping did provide a precise method of fingerprinting the organisms.
Atteirpts to genotype the bifidobacteria were unsuccessful; however, members of
the genus show promise as pollution indicators because they were found in high
concentrations in human feces and sanitary sewage. Further work to determine
the natural sources of Bi f idobacter ium species should be carried out.
- V -
COKUJSlOtJS
The results of the study showed that the levels of bacterial indicators,
particularly fecal coliforms, Escherichia coli. Pseudomonas aeruginosa . and
Bi f idobacter ium spp., were hi(^ in the high priority storm sewer line at or
near saitpling points A, B and Y. The data suggest that there is an impact near
site A in the storm sewer line that itay be due to human fecal pollution.
Characterization of the indicator bacteria by means of biochemical
testing, serotyping and genotyping produced notable results. For exanple,
speciation of the fecal streptococci was shown to be a useful means of
identifying sewer or storm sewer water content. The Streptococcus species S^
faecium tended to be equally represented in all types of sanples.
Streptococcus faecal is subsp. faecal is. on the other hand, was found with
greater frequency in sanitary and priority storm sewers than in surface runoff
and non-priority sewers. In contrast, S^ faecium subsp. casseliflavus was
almost nonexistent in sanitary sewage but was the predominant enterococcus in
non-priority storm sewer water.
Mundt's 1973 S^ faecal is studies showed that isolates from non-human
sources produced proteinization reactions in litmus milk whereas the isolates
from human feces yielded acid curds. Such a trend was not observed among the
Si faecal is species isolated in this investigation and therefore the litmus
milk reactions do not appear to be a useful criterion in source tracing. As
well, genotyping of the faecal streptococcal isolates from the sanitary sewer,
priority and non-priority storm sewers and the surface nmof f did not produce
conclusive resiiLts. A total of 64 different restriction patterns were
identified among the 192 streptococcal isolates examined and the patterns did
not show any distinctive trends.
- VI -
Fecal col i form to fecal streptococcus ratios have been proposed as a
method of estimating whether the source of pollution was from a human or
nonhuman source. The FC/FS ratios were calculated during the course of this
study but the results were inconclusive. Patios greater than 4.0, suggesting a
human source, were consistently found in the sanitary sewage but the ratios in
the remainder of the saiiples varied too widely for any assuirptions to be made.
Pseudomonas aeruginosa counts were highest in sanitary and priority storm
sewage; they reached their lowest levels in non-priority storm sewage.
Serotype 0:6 was the predominant type in all samples collected. Also evident
were serotypes 0:1, 0:10, 0:11, 0:4, 0:3 and 0:2. Ihere was a tendency for
serotype 0:10 to be found primarily in sanitary sewage, but otherwise
Pseudomonas serotyping did not appear to be applicable to source
differentiation. Genotyping P^ aeruginosa organisms, on the other hand, did
produce a more precise method of fingerprinting the isolates. For example,
the ei(^t different genotypes that were obtained from the sanitary and hii^
priority storm sever cultures were not found in the non-priority sewer and
surface water runoff isolates. Also, similar REA patterns were observed in the
P. aeruginosa isolates from storm water runoff and the corresponding storm
sewer site.
Studies in our laboratory have shown that Bi f idobacterium spp. are present
in human feces in concentrations of approximately 1 x 10^ per gram. The
results of this investigation showed that bifidobacteria counts were in the lO'^
to 10^ per lOOmL range in sanitary and hi(^ priority storm sewage and decreased
by two orders of magnitude in non-priority storm sewage. Mara and Oragui
(1983) have suggested that sorbital fermenting bifidobacteria (i.e B^
adolescentis and B^ breve) could be used as indicators of human fecal
- Vll -
pollution. Hcwever, we would suggest caution in this approach because we have
been able to isolate B^. adolescentis fran dog feces and B. breve, B^ minimum
and Bi thermo]::hilum from chicken fecal saitples. It is interesting that the
nonhuman B^ thermophilum strain was also isolated from the non-priority storm
sewer at site Z.
Although three different restriction enzymes were used to digest v^ole
cell CNA from the bifidobacteria not enough CMA was recovered and the
genotyping experiments were unsuccessful. This aspect of the investigation
merits further study.
REOMlEIlEftnCNS
1. This project shewed that source determination studies can be conducted
most effectively during periods of dry weather when there is a substantial
flow in the storm sewer line. During storm events it is difficult to
trace the source of contamination because the bacterial indicatoirs come
from a variety of sources. It is therefore recommended that source
tracing be done only under dry weather conditions.
2. The results of this investigation suggest that because S^ faecium subsp.
casseliflavus was isolated primarily frcsn surface water runoff and non-
priority storm sewers it may come solely from nonhuman sources. This
concept is supported by the fact that we have been unable to isolate the
organism from a limited number of human fecal specimens. We propose that
further studies be carried out to determine the source of this organism.
Also, that field studies be done to assess the applicability of a E^ coli
to S^ casseliflavus or a Bifidobacterium to S^ casseliflavus ratio.
3. It was apparent from this project that the serotyping of P^ aeruginosa
- Vlll -
isolates did not aid in the tracing of illegal sanitary connections in
storm sewers. It is suggested that in future studies serotyping be
combined with phage typing or genotyping to fingerprint the Pj^.ndnmnn^i?^
organisms. A probe, developed from either total chramoscaral CNA or a
specific fragment common to human strains, might allow us to hybridize
against all other strains and differentiate between human and nonhuman
fecal material. «
Ihe project results suggest that bifidobacteria show promise as pollution
indicators. Additional work should be done to determine the natural
habitat of the individual Bifidobacterium species. In addition, further
attenpts could be made to genotype the organisms.
Table of Oorttents
Page No.
INHOXJCnCN
Rationale for the Use of Selected Indicator Organisnis 1
Fecal Coliforms 3
Escherichia coli 3
Fecal Streptococci and Enterococci 5
Fecal Coliform to Fecal Streptococci Ratios 6
Pseudomonas aeruginosa 8
Bifidobacterium spp. 8
Clostridium perfrinqens " 9
Scope 9
METQKOS 10
Sampling Sites 10
Sarrple Collection 10
Bacterial Isolation and Enumeration 13
Fecal coliforms, E. coli
Fecal streptococci
Enterococci
Pseudomonas aeruginosa
Bi f idobacter ium spp.
Clostridium perfringens
BACTERIAL CHARACTERIZATICN 15
Fecal streptococci; Serotyping of Pseudomonas aeruginosa
Genotyping 15
Fecal streptococci
Ps. aeruginosa
Bifidobacteria
Page No.
RESUIirS AND DISCLJSSICN 18
1986 and 1987 Sxxrveys 18
Fecal Indicator Bacteria
Pseudomonas aeruginosa
Bifidobacteria
Clostridium perfringens
FC/FS Patio
SUMMARY 36
Summary of 1986 - 1987 Survey Results 36
1988 Surveys • 37
Summary of 1988 Survey Results 47
DISCUSSICN OF ADDrnCNAL roCOECIS 50
RKFEKhUCES 55
APFQIDIX A 60
Tables 14-69 60
APFQTOIX B 116
Corrparative Study of the Survival of Indicator
Bacterial Species by Eric Bauer
APFQIDIX C 172
The Isolation and Identification of Bifidobacteria
from Fecal and Sewage Samples by In-ja Huh
APETNDIX D _ 203
A Study of the Survival of Bifidobacteria and their
Role in Water Quality Control by Sheila Shibata
Page No.
APFQIDIX E 241
Clostridium perfrincrens and Bi f idobacter ia sp. as
Tracers in Storm Sewers by Eiric Hani
APPENDIX F 254
Characterization of Pseudomonas aeruginosa from Storm
and Sanitary Sewers by Rita Harmandayan
APPENDIX G 294
Bifidobacterium sp.
APPENDIX H 299
Fecal Strepjtococci
APPENDIX I 307
Pseudomonas aeruginosa
APPENDIX J 312
Media Preparation
UST OF FIGURES
Figure Page No.
1 Sairpling sites in Mcunt Steven storm sewer and 12
sanitary sewer lines
2 Fecal coliform levels at the storm sewer and 20
sanitary sewer sairpling sites
3 Fecal strepotococcus levels at the storm sewer 21
and sanitary sewer sairpling sites
4 Pseudononas aeruginosa levels at the stonri sewer 23
and sanitary sewer saitpling sites
5 Bifidobacteria levels at the storm sewer and 25
sanitary sewer sairpling sites
6 Clostridium perfrinqens levels at the storm sewer 26
and sanitary sewer sairpling sites
7 Percentage distribution of Streptococcus faecal is 48
subsp. faecal is among the sanitary sewer sites
(D,E,F), the priority storm sewer (A,B,C,Y), the
non-priority storm sewer (X,Z) and the storm water
runoff (P,G,R,Q,) locations
8 Percentage distribution of Streptococcus faecium subsp. 49
casseliflavus among the sanitary sewage sites (D,E,F) ,
the priority storm sewer (A,B,C,Y), the non-priority
storm sewer (X,Z) and the storm water runoff (P,G,R,Q)
locations
APPENDIX
B-1 Colony Forming Units per mL vs. Time at Room 139
Tenperature
B-2 Colony Forming Units per mL vs. Time at 140
15 degrees Celsius
B-3 E. coli CRJ/mL vs. Time at Room Tenperature 141
and 15 degrees Celsius
B-4 Pi aeruginosa CKJ/mL vs. Time at Room 142
Temperature and 15 degrees Celsius
B-5 Strep, on m-Ent CFU/mL vs. Time at Room 143
Tenperature and 15 degrees Celsius
B-6 Strep, on m-E CFU/mL vs. Time at Room 144
Tenperature and 15 degrees Celsius
B-7 Bi loncaim CFU/mL vs. Time at Room Tenperature 145
and 15 degrees Celsius
APFQJDIX
Figure Page No.
B-8 Magnification of Colony Forming Units per mL vs. 146
Time at Room Teitperature and 15 degrees Celsius
B-9 Magnification of Colony Forming Units per mL vs. 147
Time at Room Temperature and 15 degrees Celsius
B-10 Magnification of Ei coli CFU/mL vs. Time at Room 148
Teitperature and 15 degrees Celsius
B-11 Magnification of P^ aeruginosa CFU/mL vs. Time 143
at Room Tenperature and 15 degrees Celsius
B-12 Effect of 0.2 ppm Oilorine on Various Organisms 151
at a pH of 6.0
B-13 Effect of 0.4 ppm Qilorine on Various Organisms 152
at a pH of 9.0
B-14 Effect of 0.2 ppm Chlorine on Various Organisms 153
at a pH of 9.0
B-15 Effect of 0.4 ppm Oilorine on Various Organisms 154
at a pH of 9.0
C-1 Geometric mean concentration of FC, E^ coli and 188
Bifidobacteria in storm sewage
C-2 A comparison of the irecovery of Bifidobacteria 193
species from feces and sewage on two selective
media.
C-3 Percentage species of Bifidobacteria in feces, 195
sanitary and storm sewage
D-1 Dialysis membrane diffusion chamhser. 216
D-2 In vitro survival of bifidobacteria isolated 224
from sewage and E^ coli in Lake Ontario water.
D-3 In vitro survival of bifidobacteria isoalted 226
from human feces and E^ coli in Lake Ontario
water.
D-4 In vitro survival of Bifidobacterium bifidum 227
(ATDC #696) and E^ coli in lake Ontario water
APFQOIX
Figure Page No.
D-5 In vitro survival of Bi f idobacter ium breve 228
(ATCC #701) and E. coli in Lake Ontario water
D-6 In vitro survival of fecal bifidobacteria and 229
E. coli in lake Ontario Water
E-1 Geometric Mean Concentrations of Fecal 248
Coli forms, E^ coli, Bifidobacteria spp. and
Clostridium perfringens in Sanitary Sewage
during Dry Weather Survey June 10, 11
and 12, 1987
E-2 Geometric Mean Concentrations of Fecal 249
Coli forms, E^ coli. Bifidobacteria spp. and
Clostridium perfrinqens in Hii^ Priority
Storm Sewage during Dry Weather Survey
June 10, 11 and 12, 1987
E-3 A coitparison of the Geometric Mean 250
Concentrations of E^ coli, Clostridium
perfrinqens and Bifidobacteria in High
Priority and Non-Priority Storm and
Sanitary Sewage (IDG values)
E-4 A conparison of the Geometric Mean 251
Concentrations of E^, coli, Clostridium
perfrinqens and Bifidobacteria in Human,
Cat and Dog Feces (HDG values)
E-5 Percentage of Sorbitol Fermenting 252
Bifidobacteria in Feces - High Priority
and Non-Priority Storm and Sanitary Sewage
F-1 Geometric Mean Concentration of Fecal 267
Indicators in Storm Sewage
F-2 Geometric mean Concentrations of Fecal 269
Indicators in Sanitary Sewage
F-3 Percentage Serotypes of Pseudomonas 270
aeruginosa in Storm and Sanitary Sewage
F-4 Agarose gel electrophoresis of total cellular 273
CMA from Pseudomonas aeruginosa digested with
Sma I endonuclease
F-5 Agarose gel electrophoresis of total cellular 275
K^ from Pseudomonas aeruginosa digested with
SMa I endonuclease
APForoix
Figure Page No.
F-6 Agarose gel electrophoresis of total cellular 277
CNA from Pseudomonas aeruginosa digested with
SMa I endonuclease
F-7 Agarose gel electropiioresis of total cellular 284
CNA f rem Pseudomonas aeruginosa digested with
six different endonucleases
T.TfTT OF TARTry;
Table Page No.
1 Levels of E^ coli 4
2 Fecal Source Related to FC/FS Ratios 7
3 Saitpling Locations of Hi<^ Priority and 11
Non-Priority Storm Sewers, Sanitary Sewer
and Storm Water Runoff
4 Overall Geometric Mean Concentrations 19
of Fecal Coliforms, E^ coli. Fecal
Streptococci, Enterococci, Pseudomonas
aeruginosa . Bifidobacteria and
Clostridium perfringens Recovered from
Sanitary Sewage as well as High Priority
and Non-Priority Storm Sewage during Dry
and Wet Weather Surveys
5 Fecal Streptococci Populations Recovered 30
from Storm Sewers and Sanitary Sewage
during Dry Weather Survey 2, 1987
6 Percentage of S^ faecal is Isolates Producing 32
Acid Curd or Proteinization Reactions in
Litmus Milk from Dry Weather Survey 1
7 Percentage of S^ faecal is Isolates Producing 33
Acid Curd or Proteinization Reactions in
Litmus Milk from Dry Weather Survey 2 and
Wet Weather Survey 1 Samples
8 Percentage of Sorbitol Fermenting Bifidobacteria 35
in High Priority and Non-Priority Storm Sewage,
Sanitary Sewage and Feces
9 Overall Geometric Mean Concentrations of Fecal 38
Coliforms, E^ coli. Fecal Streptococci,
Enterococci, P^ aeruginosa, and Bifidobacterium sp.
Recovered from Sanitary Sewage as well as High
Priority and Non-Priority Storm Sewage during Two
Dry Weather Surveys in 1988
10 Distribution of the Prominent Pseudomonas 41
aeruginosa REA Patterns Among Sanitary Sewer,
Priority and Non-Priority Storm Sewer and Storm
Water Runoff Sairples
11 Distribution of Pseudomonas aeruginosa REA 42
Patterns Among Sanitary Sewer, Priority and
Non-Priority Storm Sewer and Storm Water
Runoff Sanples Collected during Periods of
Wet and Dry Weather
Taible Page No.
12 Distribution of Streptococcus faecal is subsp. 45
faecal is REA Patterns Among Sanitary Sewer,
Priority and Non-Priority Storm Sewer and
Storm Water Runoff Sanples Collected during
Periods of Wet and Dry Weather
13 Distribution of Streptococcus faecium subsp. 46
casseliflavus PEA Patterns Among Sanitary
Sewer, Priority and Non-Priority Storm Sewer
and Storm Water Runoff Saitples Collected
during Periods of Wet and Dry Weather
14 Saitpling Dates for Dry and Wet Weather Surveys 60
15 Geometric Mean Concentrations of Fecal 61
Coliforms, Escherichia coli. Fecal
Streptococcus, Enterococci and Pseudomonas
aeruginosa Recovered from Hi(^ Priority Storm
Sewage during Dry Weather Survey 1, 1986
16 Geometric Mean Concentrations of Fecal 62
Coliforms, Escherichia coli. Fecal
Streptococcus, Enterococci and Pseudomonas
aeruginosa Recovered fron Sanitary Sewage
during Dry Weather Survey 1, 1986
17 Geometric Mean Concentrations of Fecal 63
Coliforms, Escherichia coli. Fecal
Streptococcus, Enterococci and Pseudomonas
aeruginosa . Bifidobacteria and Clostridium
perfringens Recovered from Hi(^ Priority and
Non-Priority Storm Sewage During Dry Weather
Survey 2, 1987
18 Geometric Mean Concentrations of Fecal 64
Coliforms, Escherichia coli. Fecal
Streptococcus, Enterococci and Pseudomonas
aeruginosa. Bifidobacteria and Clostridium
perfringens Recovered from Sanitary Sewage
during Dry Weather Survey 2, 1987
19 Geometric Mean Concentrations of Fecal 65
Coliforms, Escherichia coli. Fecal
Streptococcus, Enterococci and Pseudomonas
aeruginosa. Bifidobacteria and Clostridium
perfringens Recovered from High Priority
and Non-Priority Storm Sewage during Dry
Weather Survey 2, 1987
Table Page No.
20 Geonvstric Mean Concentrations of Fecal 66
Coliforms, Escherichia coli, Fecal
Streptococcus, Enterococci and Pseudomonas
aeruginosa. Bifidobacteria and Clostridium
perfrinqens Recovered f rem Sanitary Serfage
during Dry Weather Survey 2, 1987
21 Geometric Mean Concentrations of Fecal 67
Coliforms, Escherichia coli. Fecal
Streptococcus, Enterococci and Pseudomonas
aeruginosa, Bifidctoacteria and Clostridium
perfringens Recovered from Non-Priority
Sewage and Runoffs during Wet Weather
Surveys in 1987
22 A Conparison of Fecal Coliforms/Fecal 68
Streptococcus (FC/FS) Ratios (FC:FS)
at Sairpling Sites
23 Fecal Streptococci Populations Recovered 69
from Storm Sewers and Sanitary Sewage during
Dry Weather Survey 1, October 21, 1986
24 Fecal Streptococci Populations Recovered 70
fran Storm Sewers and Sanitary Sewage during
Dry Weather Survey 1, October 22, 1986
25 Fecal Streptococci Populations Recovered 71
from Storm Sewers and Sanitary Sewage during
Dry Weather Survey 1, October 28, 1986
26 Fecal Streptococci Populations Recovered 72
from Storm Sewers and Sanitary Sewage during
Dry Weather Survey 1, November 18, 1986
27 Fecal Streptococci Populations Recovered 73
from Storm Sewers and Sanitary Sewage during
Dry Weather June 10, 1987
28 Fecal Streptococci Populations Recovered 74
from Storm Sewers and Sanitary Sewage during
Dry Weather June 11, 1987
29 Fecal Streptococci Populations Recovered 75
from Storm Sewers and Runoffs during
Wet Weather Survey 1, July 14, 1987
30 Fecal Streptococci Populations Recovered 76
fron Non-Priority Storm Sewage and Runoffs
during Wet Weather Survey, September 18, 1987
31 Fecal Streptococci Populations Recovered 77
from Storm Sewers (Non-Priority) and Runoffs
during Wet Weather Survey October 21, 1987
Table Page No.
32 Percentage of S^ faecal is Isolates Producing 78
Acid Curds or Proteinization Reactions in
Litmus Milk from Dry Weather Survey 1,
Octcber 21, 1986
33 Percentage of S^ faecal is Isolates Producing 79
Acid Curds or Proteinization Reactions in
Litmus Milk frcm Dry Weather Survey 1,
October 22, 1986
34 Percentage of S^. faecal is Isolates Producing 80
Acid Curds or Proteinization Reactions in
Litmus Milk from Dry Weather Survey 1,
October 28, 1986
35 Percentage of S^ faecal is Isolates Producing 81
Acid Curds or Proteinization Reactions in
Litmus Milk from Dry Weather Survey 1,
November 18, 1986
36 Percentage of S^. faecal is Isolates Producing 82
Acid Curds or Proteinization Reactions in
Litmus Milk from Dry Weather June 10, 1987
37 Percentage of S^ faecal is Isolates Producing 83
Acid Curds or Proteinization Reactions in
Litmus Milk from Dry Weather June 11, 1987
38 Percentage of S^ faecal is Isolates Producing 84
Acid Curds or Proteinization Reactions in
Litmus Milk frcm Wet Weather 1, July 14, 1987
39 Percentage of S^ faecal is Isolates Producing 85
Acid Curds or Proteinization Reactions in
Litmus Milk from Wet Weather Survey
September 18, 1987
40 Percentage of S^ faecal is Isolates Producing 86
Acid Curds or Proteinization Reactions in
Litmus Milk from Wet Weather Survey
October 21, 1987
41 Percentage of Serotypes of Pseudomonas 87
aeruginosa from High Priority Storm Sewers
during Dry Weather Survey October 21, 1986
42 Percentage of Serotypes of Pseudomonas 88
aeruginosa from High Priority Storm Sewers
during Dry Weather Survey October 22, 1986
43 Percentage of Serotypes of Pseudomonas 89
aeruginosa from High Priority Storm Sewers
and Sanitary Sewage during Dry Weather
Survey November 18, 1986
l^le . ■ P^ge No.
44 Percentage of Serotypes of Pseudomonas 90
aerijainosa from High Priority Storm Sewers
ard Sanitary Sewage during Dry Weather
Survey June 10, 1987
45 Percentage of Serotypes of Pseudcanonas 91
aeruginosa f rem Hi(^ Priority Storm Sewers
and Sanitary Sewage during Dry Weather
Survey June 11, 1987
46 Percentage of Serotypes of Pseudcarxpnas 92
aeruginosa from Uigin Priority Storm Sewers
and Sanitary Sewage during Dry Weather
Survey June 12, 1987
47 Percentage of Serotypes of Pseudomonas 93
aeruginosa f rem High Priority Storm Sewers
and Sanitary Sewage during Dry Weather
Survey August 17, 1987
48 Percentage of Serotypes of Pseudomonas 94
aeruginosa from High Priority Storm Sewers
during Wet Weather Siirvey 1, July 14, 1987
49 Percentage of Serotypes of Pseudomonas 95
aeruginosa from Hi^ Priority Storm Sewers
and Sanitary Sewage during Wet Weather
Survey September 18, 1987
50 Percentage of Serotypes of Pseudomonas 96
aeruginosa from High Priority Storm Sewers
and Sanitary Se^rage during Wet Weather
Survey October 21, 1987
51 Geometric Mean Concentrations of Fecal 97
Coliforms, Escherichia coli. Fecal
Streptococci, Enterococci, Pseudomonas
aeruginosa , Bifidobacteria and Clostridium
perfringens Recovered from Hi(^ Priority
Storm Sewage during Dry Weather in 1988
52 Geometric Mean Concentrations of Fecal 98
Coliforms, Escherichia coli. Fecal
Streptococci, Enterococci, Pseudomonas
aeruginosa. Bifidobacteria and Clostridium
perfringens Recovered from Sanitary Sewage
during Dry Weather Survey in 1988
53 Geometric Mean Concentrations of Fecal 99
Coliforms, Escherichia coli. Fecal
Streptococci, Enterococci, Pseudomonas
aeruginosa , Bifidobacteria and Clostridium
perfringens Recovered from Non-Priority
Storm Sewage during Dry Weather in 1988
Table Page No.
54 Percentage of Serotypes of Pseudomonas 100
aeruginosa fron High Priority Storm Sewers
and Sanitary Sewage during Dry Weather
Survey June 20, 1988
55 Percentage of Serotypes of Pseudcaronas 101
aeruginosa f rem High Priority Storm Sewers
and Sanitary Sewage during Dry Weather
Survey June 21, 1988
56 Percentage of Serotypes of Pseudomonas 102
aeruginosa from High Priority Storm Sewers
and Sanitary Sewage during Dry Weather
Survey June 28, 1988
57 Percentage of Serotypes of Pseudomonas 103
aeruginosa from High Priority Storm Sewers
and Sanitary Sewage during Dry Weather
Survey August 15, 1988
58 Percentage of Serotypes of Pseudomonas 104
aeruginosa from Hi<^ Priority Storm Sewers
and Sanitary Sewage during Dry Weather
Survey August 22, 1988
59 Fecal Str^tococci Peculations Recovered 105
from Storm Sewers and Sanitary Sewage
during EJry Weather June 20, 1988
60 Fecal Streptococci Populations Recovered 106
f rcan Storm Sewers and Sanitary Sewage
during Dry Weather June 21, 1988
61 Fecal Str^tococci Peculations Recovered . 107
from Storm Sewers and Sanitary Sewage
during Dry Weather June 28, 1988
62 Fecal Streptococci Populations Recovered 108
from Storm Sewers and Sanitary Sewage
during Dry Weather August 15, 1988
63 Fecal Streptococci Populations Recovered 109
f rem Storm Sewers and Sanitary Sewage
during Dry Weather August 22, 1988
64 Percentage of S^ faecal is Isolates 110
Producing Acid Curds or Proteinization
Reactions in Litmus Mile from Dry
Weather June 20, 1988
65 Percentage of S^ faecal is Isolates 111
Producing Acid Curtis or Proteinization
Reactions in Litmus Mile from Dry
Weather June 21, 1988
Table Page No.
66 Percentage of S^ faecal is Isolates 112
Producing Acid Curds or Proteinization
Reactions in Litmus Mile frcm E)ry
Weather June 28, 1988
67 Percentage of S^ faecal is Isolates 113
Producing Acid Curds or Proteinization
Reactions in Litmus Mile from Dry
Weather August 15, 1988
68 Percentage of S^ faecal is Isolates 114
Producing Acid Curds or Proteinization
Reactions in Litmus Mile from Dry
Weather August 22, 1988
69 Summary of the Percentage of S^ faecal is 115
Isolates Producing Acid Curds or
Proteinization Reactions in Litmus Milk
in 1988 Dry Weather Sanples
APFtNOrX
B-1 Media and Incubation Parameters for 128
Enumeration of Selected Bacterial Groups
B-2 Colony Forming Units per Millilitre 135
at Room Tenperature
B-3 Colony Forming Units per Millilitre 136
at 15 degrees Celsius
B-4 Effect of Chlorine at a e« of 6.0 150
B-5 Effect of Chlorine at a pH of 9.0 150
B-6 Effect of pH on Forms of Chlorine 160
C-1 The Carbohydrate Fermentation Profile 184
used to Speciate the Bifidobacteria
Isolated from the Fecal and Sewage
Sairples
C-2 Gecmetric Mean Concentration of PC, 187
E. coli and Bifidcbacteria in the storm
and sanitary sewage samples
C-3 Geometric Mean Concentrations of E^ coli 189
and Bifidobacteria in the feces of humans,
cats and dogs
C-4 Ihe carbohydrate fermentation reactions of 190
ATCC cultures
Table Page No.
C-5 The Biochemical Test Scheme used for the 191
Identification of Bifidobacteria in the
Profiles of ATCC Cultures and Their Gram
Stain Characteristics
C-6 Species of Bifidobacteria Isolated frxan the 192
Two Selective Media that Occurs in Fecal
and Sewage Sairples
D-1 Source of Environmental Water used in vitro 217
for the Study of Bif idobacterial Survival
D-2 Conparison of Levels of Bifidobacteria 220
Isolated from Human Feces on YN-17 and MEN
D-3 levels of Bifidobacteria and E^ coli in 221
Human Feces as Isolated on YN-17 and m-TEC
D-4 levels of Sorbitol -Fermenting Bifidobacteria 222
and Ei coli in Human Feces as Isolated on
HBSA and m-TEC
I>-5 Mean Survival Counts of Bifidobacteria and 223
E. coli in Lake Ontario Water Enumerated
Over a Maximum Three Day Period
E-1 Ratio of Ei coli to Clostridium perfrinqens 246
and of Bifidobacteria to E^ coli for High
Priority and Non-Priority Storm and
Sanitary Sewage
E-2 Geometric Mean Concentrations of Fecal 247
Coliforms, E. coli, Clostridium perfrinqens
and Bifidobacteria in High Priority,
Non-Priority Storm and Sanitary Sewage
F-1 Media and Incubation Parameters for 262
Enumerating Selected Bacterial Groups
F-2 Serotype Distribution of Pseudomonas 271
aeruginosa in Storm and Sanitary Sewage
F-3 Result of Agarose Gel Electrophoresis of 274
Total Cellular CMA Extracted From Pseudomonas
aeruginosa
F-4 Correlation of Various Serogroup Typing 280
Schemes Based on 0 Ag
F-5 Worldwide Frequency of Incidence of Serotypes 282
of Pseudomonas aeruginosa
ABBREVIAnCNS
REA - Restriction Endonuclease Analysis
FC - fecal coliforms
FS - fecal streptococcus
P. - Pseudomonas
B. - Bifidobacterium
S. - Streptococcus
E. - Escherichia
L - litre
IG - indoxyl-/?-D-glucoside
SDS - sodium dodecyl sulpiiate
EOTA - ethylenediaminetetraacetic acid
TAE - Tris base, 1.0 sodium acetate, 0.1 M disodium EDTA
CFU - colony forming units
Var - variety
ATCC - American Type Culture Collection
E - exponential
- 1 -
INIRDDUCnCN
Storm sewers were designed to channel storm water frcm urban areas into
surface waters primarily to avoid the flooding that would occur as a
consequence of the blocking of natural flew patterns that existed before
urbanization. The content of storm sewers should be similar to direct runoff
and the flew restricted primarily to storm events. A small amount of dry
weather flow is not unusual in storm sewer lines due to ground water intrusion
and human activities such as the watering of lawns. However, significant dry
weather flow coi^led with high fecal coliform counts may indicate that illegal
sanitary connections are present somev^ere in the storm sewer line. This
situation presents a health hazard since storm sewage is not normally treated
prior to entering receiving waters such as Lake Ontario beach areas or the
Humber and Don Rivers which flow into Lake Ontario near beaches.
Reports of the Toronto Area Watershed Management Study (TAWMS) in 1983 and
1984 have identified contaminated storm sewer outfalls as a major source of
bacterial pollution loadings to the lower Humber River watershed (Gartner Lee
and Assoc. 1983; Weatherbee and Novak, 1984). The 1983 outfall inventory study
of the lower Humber River basin identified ei<^ty-four dry weather outfalls
that were considered sufficiently active to warrant intensive saitpling and
testing (Gartner Lee eind Assoc. 1983) . A similar study initiated in the Don
River watershed indicated that 16% of all dry weather outfalls exceeded the
recommended guidelines for fecal coliform densities (TAWMS Tech. Rep. No. 11,
1986) .
The Ministry of the Environment has designated storm sewer outfalls
discharging more than 1 L/sec. during dry weather periods and exhibiting fecal
coliform densities of greater than 10,000 FC/lOO mL as being hi^ priority and
- 2 -
have called upon municipal agencies to identify and eliminate the source of the
fecal pollution in these sewage lines. Follow up action has been initiated on
identified outfalls in the Humber River watershed and already several illegal
cross-connections between sanitary and storm sewers have been located and
corrected (Weatherbee and Novak, 1984) . Despite some successful attempts,
compliance with the Ministry's directive can be difficult at times, due to the
fact that methods for source determination are under-developed. Escherichia
coli v^ch is an excellent indicator of fecal contamination does not lend
itself to source determination due to its wide-spread occurrence in all fecal
material. Older methods such as the ratio of fecal coliforms to fecal
streptococci (FC/FS) have been used. Hcwever, the ratios proposed by
Geldreich in 1969 can no longer be considered valid. A major reason for this
is that bacterial isolation and enumeration methods currently used in Ontario
differ significantly from those used to develop the FC/FS ratio. As well, it
has been shown that certain animal hosts; i.e. dogs, gulls and pigeons exhibit
similar ratios to that of humans (Seyfried, Harris and Young, 1986 unpublished
data) and that the ratios obtained from polluted waters change over time as a
result of environmental stress (Seyfried, Harris and Young, 1986 unpublished
data; Feachem, 1975) .
Recently, newer methods of source determination have been proposed. These
methods entail the use of a variety of indicator organisms as described below.
Rationale for the Use of Selected Indicator Organisms
The levels of pathogens in polluted water and aniinal feces are highly
variable since the densities reflect the intestinal diseases that are prevalent
in human or other animal populations at a given time. As a result, monitoring
- 3 -
of water for possible detection of waterbome pathogens requires a variety of
conplex, time-consuming, and often insensitive procedures. The use of a
bacterial indicator system that will detect and measure fecal pollution from
all warm-blooded animals is a more realistic approach.
Fecal Col i forms
The fecal col i form bacteria do have a direct correlation with fecal
contamination from warm-blooded animals. The ability to ferment lactose with
gas production at 44.5°C is the principal biochemical characteristic used to
identify fecal coliforms. Geldreich (1966) has shewn that 96.4% of the
coliforms in human feces were positive by this test. Soils that are
contaminated with fecal discharges or ejqxssed to polluted water will contain
varying levels of fecal coliforms (Van Donsel, Geldreich and Clark, 1967;
Geldreich et al . . 1968). The low levels of coliforms that have been detected
on vegetation are derived from animal manure or ni(^t-soil used as fertilizers,
or by contact with contaminated insects and agricultural pests (Geldreich,
Kenner and Kabler, 1964) . During periods of rainfall, contamination that might
be associated with vegetation could enter surface waters via storrawater
drainage (Geldreich et al . . 1968).
Escherichia coli
In Europe, there is a long standing tradition to distinguish between
total coliforms and Escherichia coli because the latter is considered to be
more significant (Leclere et al . . 1977). According to Cabelli (1977), E. coli
is the only coliform biotype that is consistently and exclusively associated
with fecal wastes of warm-blooded animals. Ihus the significance of the
_ 4 -
presence of E. coli in surface waters is that fecal contamination due to
humans or warm-blooded animals has occurred and therefore a potential health
hazard risk from microbial or viral enteric pathogens does exist. The levels
of E. coli found in various animals, birds, and sewage effluents are given in
the table below.
Table 1. Levels of E. coli
(frcan Jones and White, 1983)
Fecal production
g/d
Average number
E. coli/q faeces
Daily '.
Load
E. coli
Man
150
13 X 10^
1.9
X
109
Cow
23600
0.23 X 10^
5.4
X
109
Hog
2700
3.3 X 10^
8.9
X
109
Sheep
1130
16 X 10^
18.1
X
109
Duck
336
33 X 10^
11.1
X
109
Turkey
448
0.3 X 10^
0.13
X
109
Chicken
182
1.3 X 10^
0.24
X
109
Gull
15.3
131.2 X 10^
2
X
109
Sewage
Sewage effluent
E.
coli concentratior
3.4 X 10^ - 2.8 )
1 X 10^ - lo'
1 100 ml
{ lo'^
7
Freshwater
Seawater
E. coli survival
mean Tgo 62.3 h
mean Tgg 2.3 h
Dufour's (1977) finding that E. coli showed the best relationship to
- 5 -
gastrointestinal illness, rather than members of the KLebsiella-Enterobacter-
Citrobacter group, is not surprising in view of its position of dominance in
the distribution of coliforms found in fecal wastes from humans and other warm-
blooded animals.
Fecal Streptococci and Enterococci
Fecal streptococci are defined as those species of streptococci which are
recovered from feces in significant quantities (Clausen et al . . 1977).
The term enterococci is used to describe those species of fecal streptococci
that grow at both 10° and 45° C and in the presence of 40% bile. Growth should
occur at a pH of 9.6 and in the presence of 6.5% sodium chloride. Esculin
hydrolysis is also positive for these organisms. Ihe group D enterococci
include S. faecal is. S. faecium, S. durans. S. avium, and related biotypes
(Sergey's Manual, 1986).
The occurrence of fecal streptococci in water suggests fecal pollution and
their absence indicates little or no warm-blooded animal contamination
(Geldreich and Kenner, 1969) . Ihere is no indication that they multiply in
natural or fecally polluted waters or soils (Clausen et al . , 1977). Cabelli
(1977) has suggested that the enterococcus group most closely meets the
characteristics of an ideal indicator, particularly because it survives better
than E. coli in the aquatic environment.
A study conducted at the University of Toronto (Seyfried, Harris and Young,
1986 unpublished) found that differences in the relative prcportions and
biotypes of group D str^Jtococci existed between human and animal hosts. These
findings are also supported by previous workers (Wheater, et al. 1979, Kenner
- 6 -
1978; and Geldreich 1976). Hill et al. (1971) found that human subjects fed on
a mainly mixed western diet carried a higher percentage of S. faecal is
biotypes. Mundt (1982) also found S. faecal is to be the predominant species
of group D streptococci in humans and was able to demonstrate marked
differences in the litmus milk reactions of S. faecal is from humans, animals
and plant material (Mundt, 1973) .
Fecal Col i form to Fecal Streptococci Ratios
The FC/FS ratio has been proposed as a means of estimating v*iether the
pollution originated from a human or a nonhuman source (Geldreich, 1966;
Geldreich and Kenner, 1969, Geldreich et al. 1968). Geldreich suggests that,
"Fecal coliform bacteria are more numerous than fecal streptococci in domestic
sewage, with a fecal coliform to fecal streptococcus ratio always greater than
4.0. As mi^t be expected, similar ratios are common to the feces of man.
Conversely, fecal streptococci are more numerous than fecal coliforms in the
feces of farm animals, cats, dogs and rodents. In feces from these animals,
the fecal coliform (FC) to fecal streptococcus (FS) ratios are less than 0.7.
Similar lew ratios are corranon to urban stormwater and farmland drainage"
(Geldreich, 1972).
The obvious major weakness of this approach is that, unless the FC and FS
die off at identical rates, the FC/FS ratio will gradually change and will no
longer reflect the original ratio in the fresh fecal material. Geldreich and
Kenner (1969) made note of this fact and recommended that the PC/FS ratio was
only valid during the first 24 hours immediately following the discharge of
bacteria into the stream. However, it is not always possible to judge the age
of the pollution and, even if one can, one cannot always estimate the time
- 7 -
between excretion and discharge into the stream. As a result McFetters et
al. (1974) concluded that the FC/FS ratio "is no longer of significance in
determining the source of the contamination when considering bacteria that
originate from domestic sewage" .
IfcFetters et al. (1974) found that enterococci survive better than FC v*iich
survive better than S. bovis and S. equinus. Feachem (1975) has prcposed that
this differential die-away can in fact strengthen the value of the FC/FS ratio
as a means of distinguishing human from non-human pollution. For example, in
fecal material in v*iich enterococci are the dominant FS group (as in human
feces) the FC/FS ratio will tend to fall viiereas in fecal material in v^ch S.
bovis and S. equinus dominate (for instance in cattle and pig feces as
suggested by Deibel, 1964; McFetters et al. 1974; Raibaud et al. 1961). the
FC/FS ratio will tend to rise. A predominantly human source should exhibit an
initially high (>4) ratio v^iich should then fall v^ereas a non-human source
should ejdiibit an initially lew ratio (<0.7) v^ch should subsequently rise.
Feachem 's conclusions are summarized in the folloving table.
Table 2. Fecal source related to FC/FS ratios
Initial
FC/FS ratio
Change through
time of
FC/FS ratio
Probable fecal
source
> 4
Rise
Fall
Uncertain
Human
< 0.7
Rise
Fall
Nonhuman
Uncertain
- 8 -
Pseudomonas aeruginosa
Ihis organism is an opportunistic pathogen of man and animals v^iich may be
spread by water. Althou(^ grcwth of the organism in water may occur under
certain conditions, the major source of P. aeruginosa in waters appears to be
fecal wastes of man and of animals associated with man. Studies have shown
that there frequently appears to be little relation between populations of P.
aeruginosa and those of other pathogens or fecal indicators (Hoadley, 1977) .
Qiaracterization of Pseudomonas aeruginosa isolated from weiste-waters may
also be of significant value in source determination because this species is
found primarily in human as opposed to other domestic animal wastes; i.e. dogs
and cats (Seyfried, Harris and Young, 1986 unpublished data; Wheater et al.
1979) and because serotyping allows for sub-speciation of this organism into
approximately 17 different heat-stable somatic antigenic groups (Kusama, 1978) .
Source tracing of Pseudomonas aeruginosa infections by serotyping has been
used successfully in many clinical trials (Young and Moody, 1974; Baltimore et
al. 1974) . Ihe method has also been used to trace the source of infection in
swimmers (Seyfried and Fraser, 1978) and could possibly be adapted to tracing
studies in other environmental settings.
Bifidobacterium spp.
It has been suggested that Bifidobacterium spp. are good indicators of
human fecal wastes in surface waters (Buchanan and Gibbon, 1947; Leven, 1977;
and Resnick and Leven, 1981) . Bifidobacteria are present in concentrations of
10^ organisms per gram of feces in humans (Geldreich, 1979) but have a very
_ 9 _
limited distribution among other animals (Mara and Oragui, 1983) . Ihey have
also been recovered from raw sewage (Resnick and Leven, 1981) . Mara and
Oragui (1983) reported that sorfeitol fermenting species of bifidobacteria were
exclusive to human fecal wastes and proposed a membrane filtration method for
recovering bifidobacteria from surface waters.
Clostridium perfringens
Clostridium perfringens was suggested as an indicator of the pollution of
water with fecal wastes in the late 1890s (Cabelli, 1977) . However, because of
the organism's extreme resistance, abundance in deconposing organic matter and
soil, and failure to occur in numbers that correlate with the results of a
sanitary survey (Levine, 1921) it is rarely used as an indicator. Nonetheless,
the enumeration of C. perfringens spores as a water guality indicator appears
to have specific and limited applications, primarily as an adjunct to the
commonly used coliform and fecal streptococcus indicators.
C. perfringens is an indicator of choice v*ien the requirement is for
measuring remote and intermittent sources of pollution reaching an area (such
as, tracing sewage sludge dunped into bodies of water or the infiltration of
ground water supplies with fecal wastes) and in instances where other microbial
indicators are rapidly destroyed, such as chlorinated water supplies.
Scope ', .. •
In order to determine the relative merits of the novel source
determination methods and to devise a methodology for the detection of human
fecal wastes in storm sewer lines, a study to characterize the bacterial
populations found in urban storm and sanitary wastes was initiated in the fall
- 10 -
of 1986. Fecal indicator bacteria were enumerated in sanitary, storm and
priority storm sewage and storm water runoff. Fecal streptococci and
Pseudomonas aeruginosa were isolated and identified according to the
phenotypes, serotypes and genotypes present in the different sairple types.
Bifidobacteria were also enumerated fron storm and sanitary wastes and
recovered isolates were tested for sorbitol fermentation.
MEmODS
Sampling Sites
Sites A, B and C (shown in Table 3 and Fig. 1) in the Mount Steven Trunk
storm sewer line were sampled because this area was designated a high priority
sewer by the Ministry of the Environment. The non-priority sites, selected for
conparison, were X, Y and Z in the Mount Steven Trunk storm sewer branch lines.
Curing periods of wet weather, storm water run-off was also collected at X, Y
and Z sites. These sairples were labelled R, G. and Q, respectively. Saitples
D, E and F were obtained frcm a sanitary sewer in close proximity to the
priority storm sewer sanpling points.
Sample Collection
IXjring periods of dry weather, triplicate sanples were collected from each
sampling point in the sewers over a two to four-day period. The dry weather
sanples were obtained in October, 1986; June, 1987; August, 1987; June, 1988;
and August, 1988. Wet weather sanples were collected from the sewers and the
street run-off during rainy days in July, September and October, 1987. (See
Appendix Table 14 for precise sampling dates) .
- 11 -
Table 3. Saiipling locations of high priority and non-priority
storm sewers, sanitary sewer and storm water runoff.
Sample
Description
Code
Site
High Priority
Storm Sewer Line,
Mount Steven
Storm Sewer Trunk
B
Danf orth and Jones Avenues - furthest
in-line sanpling point (near source of
suspected solution input) .
Pape and Strathcona
saitpling point) .
Avenues (mid-line
First and Broadview Avenues (near outfall)
Non-Priority
Storm Sewer
Branch Lines
Chatham and Jones Avenues (connects to main
line above saitpling point B) .
Danf orth and Woody crest Avenues (connects to
main line above sanpling point A) .
Pape and Cavell Avenues.
Storm Water
Runoff
G
R
Q
Chatham and Jones.
Danf orth and Woodycrest Avenues
Pape and Cavell Avenues
Sanitary
Sewage Line
D
E
F
Danf orth and Jones Avenues
Strathcona and Pape Avenues
First and Broadview Avenues
- 12 -
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- 13 -
The saiiples were collected in sterile glass containers, transported to the
laboratory on ice, and processed within 6 hours of collection.
Fecal specimens fran both humans and amirals were also obtained.
Bacterial Isolation and Enumeration
Analysis of the sairples for indicator bacteria was by membrane filtration
of appropriate dilutions of the saitples through Gelman GN6 47 ram cellulose
nitrate filters with a pore size of 0.45 um (Standard Methods, 1985). The
filters were planted on media appropriate for the recovery of the various
indicator bacteria.
Fecal coliform bacterial densities were determined by planting the filters
on m-TEC agar (Dufour et al. 1981) and incubating for 23 ± 1 hours at 44.5 +
0.5° C. Both target and non-target colonies were counted. Target colonies
were yellcw, yellcw-green and yellcw-brcwn; non-target colonies were blue to
blue-green in colour. To ensure the accuracy of the counts, only results
obtained from filters with target counts between 10 and 100 colonies were used
to calculate the bacterial density per 100 mL of water sanple.
A second step for the determination of E. coli by urease treatment (Cufour
and Cabelli, 1975) was incorporated into the m-TEC procedure. Filters with
appropriate target counts (i.e. between 10 and 100 target colonies) were
removed from the m-TEC plates and placed on filter pads soaked in a urea phenol
red solution (Dufour and Cabelli, 1975) . The filters remained in contact with
the filter pad for 15 min. to allcw for deaminization by non-E. coli coliform
bacteria processing urease. A second count of all urease-negative colonies
(all yellow, yellcw-green and yellcw-brown colonies) was taken.
Fecal streptococci determinations were made by planting the filters on m-
- 14 -
Enteroccxxnjs agar (Difcx>) (AHiA, 1985) . The medium was incubated for 48 hr.
at 35 °C and a ccfunt of all pink to purple target colonies taken. Upper and
Icwer counting limits of 10 to 150 target organisms were applied to the
reported results.
Enterococci were recovered on m-ME agar (AEHA, 1985) v*iich also contains
indoxyl-B-D-glucoside (IG) . The m-ME plates were incubated for 48 hours at
41.5 ± 0.5° C. The incubation time was modified from the original 24 hr.
suggested by Dufour as it allowed for a slight increase in recovery of target
organisms. Ihe addition of IG to the medium facilitates differentiation of the
B-D-glucosidase enterococci from other fecal streptococci. Both target and
non-target colonies were enumerated. Targeted colonies on m-^1E were purple,
v*iite-blue to dark blue with blue haloes from degradation of the IG. Non-
targets were pink to maroon non-haloed colonies. Counting range limits of 10
and 150 were also applied to these results.
Pseudomonas aeruginosa densities were determined using m-PA agar (AHiA,
1985) . The medium was incubated at 41.5 + 0.5° C for 48 hr. and a count of
all flat spreading brcwnish-green or tan colonies obtained. Upper and lower
counting limits of 10 and 150 were afplied to the results.
Bi f idobacter ium spp. were isolated on the YN-17 medium described by Mara
and Oragui (1983) . Target bifidobacteria colonies appear dark blue to black.
Greenish coloured colonies were not counted; however, to ensure that all
bifidobacteria were enumerated, crystal violet stains were made of colonies
which had a different size or colour. Adjustments to the count were made if
necessary.
Clostridium perfrinaens was isolated using a medium originally developed
by Bisson and Cabelli (1979) and modified by the Ministry of the Environment
- 15 -
Southeastern Region Laboratory (in-CP2) (M.O.E., 1986). The sanples were
pretreated at 70° C for 15 or 30 min. to destroy the vegetative cells.
Appropriate volumes of the sairples were passed thrtxK^ membrane filters and the
filters were placed on m-CP2 plates. The plates were then incubated
anaerobically at 37° C for 48 hr. Target colonies appear yellcw with a large
black centre v*iich can extend fairly close to the circumference; the colonies
do not possess a blue halo. Nontarget colonies have a blue halo and they can
be yellow or yellcw with a black centre.
Formulations for the aforementioned media are provided in the Appendix.
All bacteria were identified to the species level using standard taxonomic
methods.
Bacterial Characterization . . ..
Approximately 2,500 fecal streptococci were recovered on m-Enterococcus
agar and m-ME agar during the dry and wet weather surveys. The isolates that
were identified as varieties of S. faecal is were tested for their reaction in
litmus milk broth (Difco) using the method of Mundt (1973) .
Serotyping of the P. aeruginosa isolates was carried out using a
Pseudomonas Antisera Kit (Difco) . The organisms were sub-speciated into the 17
different heat-stable somatic antigen groips described by Kusama (1978) .
Genotypinq
For the restriction enzyme analysis (REA) of P. aeruginosa, total cellular
raJA was extracted using a method described by Bradbury et al. (1984, 1985) . A
1.5 mL volume of an 18 hour nutrient broth culture inoculated with P.
aeruginosa was transferred into an Eppendorf tube and centrifuged in a
- 16 -
Microfuge 12 (Beckman) for 3 minutes at 7500 x g. The supernatant was
discarded and the pellet loosened by vortexing. A 291 uL volume of PEB I
buffer containing 10 mg/mL lysozyme was added and the mixture incubated for 20
minutes at 35° C. A 9 uL amount of 5M NaCl was added, thorou^ily mixed, and
then 150 uL of 10% sodium dodecyl sulfate (SDS) was added. The solution was
gently mixed and incubated for 10 minutes at 37° C. Following the addition of
450 uL phenol: chloroform risoarayl (25:24:1), the mixture was vortexed and
centrifuged at 7500 x g for six minutes at room tenperature. The upper aqueous
phase was removed with a pasteur pipette and transferred to an Eppendorf tube.
One mL of 95% cold ethanol was added, the tubes vigorously shaken and stored at
-20° C ovemi(^t. The mixture was centrifuged for 3 minutes at 12, 000 x g,
the si.;pematant discarded and the pellet redissolved in 250 mL CNA wash buffer.
A total of 500 uL of 95% cold ethanol was added, the mixture stored at -20° C
for 20 minutes, and centrifuged at 1,200 x g for 3 minutes. The supernatant
was discarded and the pellet allowed to dry at 37° C for 10 minutes. The
pellet was dissolved in 100 uL of distilled water and stored at 4° C until
digested.
Restriction digests were performed using Sma I according to the
manufacturer's instructions (Boerhinger Mannheim). A 10 uL aliquot of double
strength (2X) Sma I buffer was placed in an Eppendorf tube and 10 uL extracted
CWA added. A 2 uL sanple of Sma I enzyme was added and the mixture was
incubated for 1 hour at 37° C to allow for ccsiplete digestion. Following the
addition of 1 uL of 0.15M EETA + 0.4 mg/mL RNase A, the tube was incubated at
37° C for 20 minutes. A 5 uL volume of 5X sairple buffer was then added to the
restriction digest. Saitples were electroj±ioresed on 0.7% agarose gel for 16
hours at 27 volts. Gels were stained with 1 mg/mL ethidium bromide in IX TAE
- 17 -
(Tris base, 1.0 sodium acetate, 0.1 M disodium EDIA) for 1 hour and destained
for 2 hours in distilled water. Hiotography was done using U.V. lii^t at 300
nm and a red No. 23A polaroid 66p/N film with an ejqxjsure time of 30 seconds.
Fecal streptococci were grcwn in Brain Heart Infusion broth (Difco) at 37°
C for 4 hours and the total cellular CNA extracted as described previcJusly.
Restriction digests were performed using Bam HI enzyme according to the
manufacturer's directions (Boehringer Mannheim) along with a 10 liL aliquot of
2X Bam HI buffer.
Bifidobacterium sp. were grcwn anaercbically in MRS broth (Oxoid) for 48
hours at 37° C. The same method as was used for P. aeruginosa and fecal
streptococci was enployed to extract total cellular DMA from Bifidotecterium
spp. When difficulties were experienced with the cellular DNA extraction
procedure, the follcwing treatments were added:
1. hi^ concentration of lysozyme (i.e. 20 mg/mL) ;
2. sodium dodecyl sulfate (SDS) with normal lysozyme concentration;
3. alkaline lysis;
4. SDS with proteinase K;
5. proteinase K without SDS;
6. Triton X-100;
7. 4M guanidine isothiocynate ; and
8 . lysostaphase .
Three different enzymes, Sma I, Bam HI and Cfo, with their appropriate
buffers, were used for the restriction enzyme analysis of the bifidobacteria.
- 18 -
RESUIJS AND DISCUSSICN
1986 and 198 7 Surveys
Ihe concentrations of fecal indicator bacteria and Pseudcmionas aeruginosa
found in sanitary s«^rage, hi<^ priority and non-priority storm sewage, and
storm water runoff are presented in Tables 15 to 21 (Appendix) . The results
summarized in Table 4 shew that the concentrations of fecal indicator bacteria
in the Mount Steven Storm Trunk were hi<^ during both dry weather surveys.
Although levels of greater than 10,000 fecal coliforms per 100 mL occurred
throughout the main line (saitpling points A, B, and C) high densities were
noted most frequently at sites A and B. The source of the suspected fecal
contamination is thought to be near site A, but the hi(^ fecal coliform and E.
coli levels exhibited in sanple B suggest that a second contaminant input may
occur somewhere in-line between points A and B. The concentrations of fecal
coliforms, and more specifically of E^ coli. in saiiple C (near the outfall)
tended to fluctuate but were usually Icwer than at sites A ard B. In
conparison, as shewn in Figs. 2 and 3, the concentrations of fecal indicator
bacteria in sanitary sewage were consistently high, as would be expected in
this type of sarrple.
As nay be seen in Table 22 (Appendix) , the fecal coliform to fecal
streptococcus ratios observed in the sanitary sewage sanples in dry weather
were generally above 4, indicating human fecal input. Ihe FC/FS ratios varied
from day-to-day in the storm sewer line, but there was a tendency tcward ratios
of > 4.0 at points A and B. At site C, the FC/FS ratios were always below 4
which would suggest that no source of contamination is present and that the
area is being iitpacted upon by upstream pollution.
- 19 -
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Pseudcmonas aeruginosa was also present in high concentrations in the
sanitary sewer (Fig. 4) . Althoijgh this organism was found to occur in only 12%
of the human population (Sutter et al. . 1967), it is consistently isolated from
sewage and highly polluted surface waters (Bonde, 1963; Hoadley, 1967; Cabelli,
Kennedy and Levin, 1976) .
Pseudomonas aeruginosa was recovered from the high priority storm sewer
(A, B and C) during all dry weather surveys in 1986 and 1987 (Tables 15 to 20
Appendix) . The levels ejdiibited during the second dry weather survey were
hi^er than those recovered in survey 1 (Table 4) possibly due to the fact that
the second survey was conducted during the summer period as opposed to the late
fall sairpling of survey 1. Pseudomonas has been shown to exhibit regrowth in
nutrient enriched waters at hi^ tenperatures (Hoadley, 1977) .
Previous investigators have shewn P^. aeruginosa to be more indicative of
human rather than animal fecal wastes (Wheater et al . , 1978, 1979). The
concentrations present at Points A and B, particularly during dry weather
survey 2, would suggest that the contaminate irput at these sites is human in
origin. Levels of Pseudomonas at point C tended to be lower than at points A
and B, again confirming that the source of the contamination lies i^sstream of
this point.
Pseudomonas aeruginosa was not isolated from the high priority storm sewer
during the first dry weather survey on October 28. Rainfall on the previous
evening may have caused dilution effect in the sewer such that P^ aeruginosa
could not be detected. Any incoming fecal material from street runoff would be
of non-human origin (e.g. dog) (Geldreich, 1979) . Ihis would cause an increase
in the fecal coliform, E^ coli and fecal streptococcus concentrations in the
sewer but would not necessarily increase P^. aeruginosa concentrations since
- 23 -
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- 24 -
animal feces (i.e. dcgs, cats, raccoons) generally do not contain Pseudomonas
(Seyfried, Harris and Young, manuscript in pr^>aration) .
Bifidobacteria was recovered frcm both hi^ priority storm and sanitary
sewage during the dry weather surveys (Tables 15 to 20, Appendix) . Ihis
organism is thought to occur primarily in human wastes (Mara and Oragui, 1983)
and was found in concentrations of 10^ to 10-^° in the human feces analyzed as a
separate facet in this study. The hi^ concentrations of this bacterium in the
storm sewer at points A and B and the Icwer concentrations found at point C
(Fig. 5) again confirms that human fecal input occurs at the two upstream
points because this organism exhibits rapid die-off in surface waters (Oragui,
1982) . .
Clostridium perfringens was included as a parameter during the second dry
weather survey. Current literature available on the bacterium suggests that it
is associated with both human and animal wastes (Bisson and Cabelli, 1980;
Geldreich, 1979) . Generally the organism is regarded as a good indicator of
fecal pollution in situations v*iere there has been environmental stress due to
disinfection, prolonged transit time or the presence of toxic wastes (Bisson
and Cabelli, 1980; Geldreich, 1979; Fujioka and Shizuraura, 1985). C^
perfringens was recovered from both high priority storm and sanitary sewage,
and was present in higher concentrations in sanitary sewage (Fig. 6) although
it could not be isolated from the human fecal sanples assayed. According to
Geldreich (1979) , C^ perfringens is found in concentrations of 10^ to 10^ per
gram of human feces but is only present in 13 to 35 percent of the population.
Additional sairples X and Y analyzed during the second dry weather survey
were collected from branch lines connecting to the main trunk line (see Table 3
and Fig. 1) . Ihese lines were thought to be non-priority storm sewage
- 25 -
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according to the City of Toronto Public Works Department. Sairple X e>diibited
low fecal indicator counts as would be ej^^ected in non-priority storm sewage.
The sanple also contained very lew Pseudcanonas and Bifidobacterium levels
v^iich woiild indicate the absence of human feccil input at this site. Sample Y,
however, e>±Libited hi(^ concentrations of fecal col i forms and E^ coli, and on
June 12 the FC counts were greater than 10,000/100 mL. Concentrations of
Pseudomonas aeruginosa and Bi f idobacter ium were also high in this sairple
suggesting that a contaminant irput, possibly of human origin is impacting on
the sewer at or near this site. Although site Y is located upstream of site B,
(see Fig. 1) it cannot account for the magnitude of pollution exhibited at B
since the bacterial concentrations recovered at Y were lower than those
presentat the B site. Because of its high fecal indicator bacterial levels,
sanple Y was groi53ed with the high priority storm sewage.
The FC/Fs ratios exhibited in sattples X and Y were generally belcw 4.
This would appear to contradict the evidence of human fecal input at site Y,
i.e. Bifidobacteria concentrations. Previous studies however, have shown that
FC/FS ratios tend to fluctuate over time as a result of the different die-off
rates of the two bacterial grotps (McNeil, 1985). It may be that the fecal
pollution irput inpacting on the sewer at site Y is located in-line above this
sanpling site. Since Bifidobacteria also tend to die-off rapidly (Oragui,
1982) , it would be interesting to determine if samples taken above site Y in
this branch line exhibited an increase in this organism cis well as an increase
in the FC/FS ration and levels of other fecal indicator bacteria.
Fecal indicator bacterial concentrations recovered from non-priority storm
sewage and storm water runoff during wet weather (Table 4) revealed that
street runoff is heavily contaminated with fecal material and contributed
- 28 -
greatly to the cnntamination in storm sewer lines during storm events. The
rainfall for this event commenced on the afternoon of July 13 and continued
until the late morning of July 14. Sanples Q, X and Z were collected after the
first flush had occurred. Sanple P was collected from street water runoff at
Parliament and Carlton streets at the start of the rainfall event and this may
account for the higher Pseudomonas levels exhibited in this storm water runoff
sanple over that of storm water sanple Q. As well, the fact that sample P was
collected from • a different geographical location may account for this
difference .
Allen Gardens is an area v4iere gulls and pigeons feed and both of these
bird species were found to carry Pseudomonas (Sey fried, Harris and Young, 1986,
unpublished data) . During wet weather, Pseudomonas aeruginosa concentrations
in storm water and storm sewage may cLLso be increased by runoff frcm vegetation
(Hoadley, 1977) .
The fecal coliform to fecal streptococci ratios eidiibited in storm water
and storm sewage during wet survey 1, were all below 4,0 suggesting fecal input
of non-human origin. It would appear that FC/FS ratios may still be a useful
diagnostic tool for source determination in storm sewer lines when applied
close to the irput, although many investigators cautioned its use in surface
water analyses (Wheater et al . . 1979; Palmer 1984; Diebel 1964).
Bifidobacteria were recovered from non-priority storm sewage samples X and
Z during wet weather. Concentrations exhibited in sanple X were much higher
than those recovered during dry weather at this site. Our studies shewed that
animals such as dogs can carry Bifidobacteria in their feces.
The distribution of fecal streptococcal populations recovered from storm
and sanitary sewage during the dry and wet weather surveys is given in Tables
23 to 31 (Appendix) . The results from dry weather survey 2 are summarized in
- 29 -
Table 5. Streptococcus faecium comprised the greatest percentage of the fecal
streptococcal group found in storm sewage and sanitary sewage during dry
weather with the exception mainly of sairples B arri F on a few sairpling days.
Seyfried, Harris and Young (1986 unpublished data) found S^ faecium to be
predominant in human fecal saitples as well as several animal species (i.e.
dogs, raccoons). Studies by Wheater et al. (1979) have also shown S^ faecium
to predominate in human feces and sanitary sewage. However, other workers
(Cooper and Ramadan, 1955; Kenner, 1978; Kjellander, 1960; and Mundt, 1982)
have found a greater percentage of S^ faecal is varieties. Dietary differences
have been shown to account for variations in the streptococcal flora of the
human intestinal tract (Hill et al . . 1971; Finegold et al . . 1975). There is a
tendency for S^ faecium to survive for longer periods than S^ faecal is in
polluted waters (Dufour 1985 personal communication) which may account for its
greater recovery from storm and sanitary sewage. Within the S^ faecal is group
there was some variation in recovery of S^. faecal is yar. faecal is and S.
faecal is var. liquefaciens in the storm and sanitary sewage saitples, with both
varieties predominating during different dry weather surveys and at different
sanple points in both sewers. Other workers have found S^ faecal is var.
faecal is to be dcminant in human feces (Kenner, 1978) . Geldreich (1979) found
that Si faecal is var. liquefaciens was present in only 26 percent of the human
population and could be recovered from environmental sources. Site X (non-
priority) ejdiibited lew percentage of Sj. faecal is varieties and showed a hi(^er
percentage of S^ faecium var. casseliflavus an organism which is found on
vegetation (Mundt and Graham, 1968) and in the feces of animals such as geese
(Seyfried, Harris and Young, 1986, unpublished data) . S^ faecium var.
casseliflavus was also recovered in high percentages in storm water and storm
- 30 -
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sewage cJuring wet weather but was generally not recovered from sanitary sewage
and has not been shown to occur in human feces (Seyfried, Harris, and Young,
1986 unpublished data) . Other str^Dtococci species not recovered in sanitary
sewage but found in storm sewage were S^ bovis and S^ avium. Both of these
species are fourd in animals such as dogs but not in human fecal material
(Seyfried, Harris and Young, 1986 unpublished data) .
The litmus milk reactions of S^ faecal is varieties summarized in Tables
6 and 7 (from Tables 32 to 40, Appendix) demonstrate that acid curd producing
strains are common in sanitary sewage. Mundt (1973) found that over 90% of S^
faecal is cultures from non-human sources such as animals, plants and insects
gave proteinization reactions in litmus milk, v^iile isolates from human feces
produced an acid curd. Seyfried et al. (unpublished data) also found that
isolates of S^ faecal is from humans did not proteinize litmus milk. However,
these same isolates also did not demonstrate the ability to produce acid curd
reactions .
Most of the isolates from sanitary sewage giving proteinization reactions
were S^ faecal is var. liquefaciens and were probably of non-human origin
because this variety can occur on such environmental sources as plants (Mundt
et al . . 1959). Isolates from high priority storm sewage shewed a hi(^
percentage of acid curd production, but this reaction was also given by some of
the non-priority storm sewage isolates from sanple X during dry and wet weather
and by isolates obtained from storm water sample Q. Although storm water and
non-priority storm sewage S^ faecal is isolates will have to be tested before
the usefulness of this test can be assessed, it would appear that the litmus
milk reactions are too nonspecific to be of value.
- 32 -
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- 34 -
The greatest percentage of Pseudoroonas aeruginosa serotypes in hi^
priority and non-priority storm sewage, sanitary sewage and storm water runoff
were serotype number 6 (Tables 41 to 50, Appendix) . Other serotypes comnvon to
all sanple types were serotypes 1, 10, 11, 4, 3, and 2. Previous research has
shewn roost of these isolates to be common to both humans and animals (Habs
1957; Sandvik 1960; and Verder and Evans 1961). On occasion, serotype 10 was
found to be more prevalent in storm sewer sample Y than was serotype 6. This
serotype was rarely isolated from non-priority storm sewage and storm water
runoff and was isolated from sanitary sewage but not to the degree that would
be expected if this serotype was coiranon to fecal material. Overall, the value
of Pseudomonas serotyping as a means of pollution source differentiation
appears to be limited and perhaps it would be more useful to look at
Pseudomonas concentrations in storm sewage.
The percentage of sorbitol fermenting Bifidobacteria in human feces and
sanitary sewage is high (Table 8). Mara and Oragui (1983) have found sorbitol
fermenting species of Bifidotacteria, i.e. B^. adolescentis and B^ breve to be
exclusive to human feces. Other workers (McNeil 1985) have also reported this
fact. The presence of these organisms in human feces is not affected by diet
and geographical variation (Drasor 1974) as are group D streptococci.
Bifidobacteria isolates obtained from dog and cat feces did not ferment
sorbitol. Although some of the non-priority storm sewage isolates gave
sorbitol fermentation reactions not enough isolates were tested (i.e. saitple
X) to make any definite conclusions about the results. Isolates from high
priority storm sewage samples A and B showed somev>^iat higher percentages of
sorbitol fermentation reactions. Sanple Y, v*iich was originally submitted as
non-priority storm sewage showed a hii^er percentage of the sorbitol
- 35 -
T^tole 8
Percentage of Scaijitol Fermenting Bifidabacteria in Hi^ Priority
and Non-Priority Storm Sewage, Sanitary Sewage and Feoes
Sanple
Souroe
Total Isolates
Identified
Number of
Sorbitol
Fermentors
D,E,F
Sanitary Sewage
A
Hi(^ Priority
B
Storm Sewage
C
Y
X
Non-Priority
Z
Storm Sevage
Feces
Human
Dog
Cat
37
35
29
12
32
15
5
38
24
18
24(65)
8(23)
4(14)
0
13(41)
3(20)
2(40)
23(61)
0
0
( ) percentage
- 36 -
fermenting strains than hi^ priroity storm sewage samples A and B and as
previously mentioned, this site could possibly be inpacted on by a human
sanitary input.
It would appear, based on these results, that sortiitol fermenting
Bifidobacteria may be good indicators of human fecal contamination.
SUmARY
Summary of 1986-1987 Survey Results
1. Fecal contamination, most likely of human origin, is present in the hi^
priority storm sewer line at or near sampling points A, B and Y.
2. Upstream contamination is impacting on the storm sewer at the downstream
area C.
3 . Street runoff during wet weather is highly contaminated with fecal
material.
4. Bifidobacteria may be useful as indicators of human fecal wastes in storm
sewage.
5. A high percentage of acid curd producing strains of S^ faecal is may be
indicative of sanitary wastes in storm sewer lines but the results are
inconclusive.
6. FC/FS ratios may be useful as a supplementary interpretive tool for source
differentiation within storm sewer lines. Ihis would have to be
investigated further because the results were not consistent at all
points.
7. Hi(^ Pseudomonas aeruginosa concentrations in storm sewage may indicate
the presence of human sanitary wastes but Pseudomonas serotyping is not
applicable to source differentiation.
8. Source determination during storm events cannot be acconplished with a
- 37 -
hi^ degree of accuracy.
1988 Surveys
Data frcm the 1986-1987 surveys (Seyfried et al . . 1987) suggested that
fecal contamination, possibly human in origin, was evident in the storm sewer
line near sites A and Y. The results of the 1988 surveys, presented in Tables
51 to 53 (Appendix) and summarized in Table 9, add support to this conclusion.
As may be seen in the table, the fecal coliform levels in the hi^-priority
storm sewer were greater than 10,000/100 mL at site A in June and at all sites
in August. It should be noted that counts of all indicator organisms tended to
be hi^er in August, possibly due to regrcwth of the bacteria in the warmer
nutrient-enriched waters (Hoadley, 1977) .
As mi^t be expected, fecal coliform and fecal streptococcus counts were
highest in sanitary sewage and the fecal coliform to fecal streptococcus ratio
was greater than 4 in these sairples (see Table 22, ^pendix) . Although a ratio
greater than 4 was observed at site A in the priority storm sewer, at other
storm sewer sites the FC/FS ratios were generally belcw 4 suggesting that there
was little or no human fecal input (Geldreich and Kenner, 1969) .
P. aeruginosa and Bi f idobacterium sp. were shown in the 1987 study to have
potential as indicators of human fecal waste. Collaborative data presented in
Table 9 shows that counts of both organisms were higher in sanitary and
priority storm sewage than in non-priority storm sewage.
Mara and Oragui first proposed the use of sorbitol fermenting
bifidobacteria as indicators of human fecal pollution in 1983. Kator and
Rhodes (1988) also used these organisms to differentiate human from animal
sources of pollution in shellfish growing waters. The species of
Bifidobacterium that are reportedly human specific and sorbitol fermenting are
g g2
- 38 -
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- 39 -
B. adolescentis and B^ breve . In this study we were able to isolate B^ breve
and Bj_ bifidum from human fecal material ; however, we also recovered B^ adolescentis
from dog fecal samples and B^. breve. B. miniinum and B^ thermophilum from chicken
feces. Twenty-one isolates that were thought to be Bi f idobacter ium on the
basis of their morphology were recovered from eight different sewage samples.
Of the 21 isolates, only two could be identified by biochemical testing. The
two were found in the non-priority storm sewer at site Z and were classified as
mannose + and mannose - strains of B^ thermophilum.
Based upon our prior use of restriction enzyme analysis to distinguish
between different strains of Klebsiella pneumoniae (Seyfried et al . . 1989), it
was felt that genotyping might assist in determining the source of the Bifidobac-
terium strains under investigation. Three enzymes were used to digest v/hole
cell IXIA from B^ adolescentis and two Bif idobacter ium sp. isolated from chicken
feces. Restriction (REA) patterns using the total cellular n^ restriction
enzyme analysis were obtained. However, \jhen we attempted to repeat the restriction
enzyme analysis, using the three different enzymes and eight modifications of
the procedure (as described in the Methods section) , we were unable to obtain
satisfactory REA patterns. It is probable that isolates from the YN-17 medium,
thought to be Bi f idobacter ium sp. , were actually streptococci because of the
initial confusion concerning the colony description (see Discussion in the
report by In-ja Huh in the Appendix) .
Similar to the 1986 and 1987 survey results, serotyping of the P. aeruginosa
1988 survey isolates showed that serotype 6 predominated in the priority and
non-priority storm sewage, sanitary sewage and storm water runoff (Tables 54 to
58, Appendix). Serotypes 1, 10, 11, 4, 3 and 2, although not as prevalent as
6, were also common in all categories of samples.
- 40 -
Seventy-ei(^t strains of P^ aeruginosa frcm the sanitary sewer and 112
strains recovered from the storm sewer and storm water runoff were also
genotyped. Forty-six different REA patterns were noted among the 191 isolates.
As may be seen from Table 10, there was an interesting distribution of patterns
among the sample groi^js. For exanple, REA patterns 1', 6', 6' and 13' were
found among isolates from the three sampling sites in the sanitary sewer. The
fact that these same patterns or genotypes were also prevalent in the priority
storm sewer samples suggests that they may be typical of himian fecal isolates.
A comparison of the corresponding serotypes for each genotype showed that the
serotypes tended to be widely distributed. For exaitple, the 1' genotype was
found in serotypses 1, 6 and 10; REA pattern 6 was distributed among serotypes
1, 3, 4, 6 and 11; 6' occurred in serotypes 1, 6, 9, 10 and 11; and the 13'
genotype was found in serotypes 6 and 10. Because it seemed lonusual to find
such a hi(^ number of genotype 6 isolates in the non-priority storm and runoff
saitples, the source of these organisms was examined. It was found that the
bacteria were all isolated during the wet weather sampling in Jvily, 1987 from
sites X and P. Ihe P site was an additional street runoff sample taken at the
beginning of the rainfall event. Ihese isolates, belonging to REA pattern 6,
were evenly divided between seroytpes 1 and 6.
Additional information provided in Table 11 shows that genotypes 1, 2, 4,
7, 9, 11", 12 and 14 were all isolated from sanitary and priority storm sewers
and not from non-priority storm sewers or storm water runoff.
In conparison P. aeruginosa isolates with genotypes 18 and 20 were found
solely in the non-priority sewer and storm runoff sairples. The REA pattern 18
organisms were distributed between serotypes 3 and 6, v*iereas pattern 20 was
found in serotypes 1 and 3. Serotype 3, REA pattern 18 P^ aeruginosa isolates
- 41 -
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- 44 -
were found in the storm water runoff sanples at site Q, and in the sewer that
collected this same storm water at site Z. Similarly, REA pattern 20 isolates
were recovered frcan water runoff at site R and in the receiving storm sewer at
site X. The fact that the genotype 20 organisms belonged to both serotypes 1
and 3 suggests that genotyping is probably a more concise method of
fingerprinting P^ aeruginosa than serotyping. From the results, genotyping
appears to be a promising method of tracing P^ aeruginosa from human and animal
sources.
Similar to the 1986-87 streptococcal results, acid curd production was
observed among the sanitary sewage and high priority storm sewage S^ faecal is
isolates (Table 64 to 69, Appendix) . However, there did not appear to be a
marked difference between the aforementioned isolates and those from the non-
priority storm sewers. All S^ faecal is isolates tended to produce a variety of
reactions in litmus milk.
Unlike the P^ aeruginosa organisms, genotyping of the fecal streptococci
did not produce any concise results. One hundred and ninety-two streptococcal
isolates, 60 of which were frcm the sanitary sewer, were genotyped. A total of
64 different REA patterns were identified among the isolates.
Streptococcus faecal is subsp. faecal is (Table 12) was the only species
that had the predominant genotypes 5 and 8 occurring in both the sanitary sewer
and the priority storm sewer isolates. As stated previously Mundt (1973) has
suggested that S^. faecal is isolates from human feces will produce an acid curd
in litmus milk. Hcwever, no relationship between the 5 and 8 pattern isolates
and those that produced an acid curd was observed. Although the S^ faecium
isolates were distributed among 31 different REA patterns, genotype 9 was found
in all three sites of the sanitary sewer and genotype 5 was recovered from all
sites in the priority storm sewer. S^ faecium subsp. casseliflavus (Table 13)
- 45 -
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- 47 -
had 21 different REA patterns that were widely distributed among the isolates
from different sources. In general, no relationship between the source
of isolation and the genotype could be found among the 192 streptococcal
isolates studied.
More insi(^t into the origin of fecal wastes in storm sewer lines was
provided by the speciation of fecal streptococci (Tables 59 to 63 , Appendix) .
The results showed that S^ faecium tended to be equally represented in all
sanple categories. On the other hand, S^ faecal is subsp. faecal is (Fig. 7) was
found more frequently in sanitary and priority storm sewers than in surface
runoff and non-priority sewers. In contrast, S^ faeccium subsp. casseliflavus
(Fig. 8) predominated in non-priority storm s&/jer water and was notably evident
in storm water runoff. The organism was virtually nonexistent in sanitary
sewage. Levels of S^ faecium subs, casseliflavus in priority storm sewers were
intermediate between the counts found in sanitary sewage and in the non-
priority storm sewer. These results concur with our previous data (Sey fried,
Harris, Young, 1986 uipublished) \A*iich showed that S^ faecium subsp.
casseliflavus could be isolated from animal feces but not from human fecal
specimens.
Summary of 1988 Survey Results
1. The levels of fecal coliforms, Escherichia coli. P^ aeruginosa and
Bi f idobacter ium sp. suggest that there is an irrpact near site A in the
storm sewer line that may be due to human feccil pollution.
2. While Bi f idobacter ium breve and B^ adolescentis are found in human feces,
they cannot be used to differentiate human from animal sources of
pollution because they can be isolated from animals such as dogs and
chickens.
- 48 -
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3. Genotyping of Bifidobacterium isolates may provide a more precise method
of source differentiation, but further work has to be done to find a
suitable genotyping method.
4. p^ aeruginosa genotyping may be of value in tracing sources of pollution.
Serotyping, however does not provide results that are specific enough.
5. Genotyping of fecal streptococci is not reccanmended as a method of source
determination since the wide variety of patterns produced yield
inconclusive results.
6. Speciating fecal streptococci is a useful means of characterizing sewer or
storm water content. For exaiiple, S^ faecal is subs, faecal is is found
predominantly in sanitary and priority storm sewers whereas S^ faecium
subsp. casseliflavus has not been isolated from human feces.
DISCUSSICN OF ADOmCNAL FRUBCES
A. Gcnparative Stixay of the Survival of Indicator Bacterial Species by Eric
Bauer (see Appendix)
Ihe conplete project is outline in the Appendix. The study had two
objectives. One was to conpare the die-off rates of E^ coli, P^. aeruginosa , S^
faecal is var. faecal is. S^ faecium and B^ longum at room tenperature and at
15° C. The second was to examine the lethality of chlorine on Ei coli. P^
aeruginosa . S. faecium var. casseliflavus and B^. breve.
The results of the project showed that B^ longum died off more quickly
than the other organisms at room tenperature. However, at 15° C, the usual
tenperature in a storm sewer, P^ aeruginosa. E^. coli. and B^. longum all had a
shorter life span than the streptococci. Thus these three organisms would be
better indicators of recent pollution in storm sewers than fecal streptococci.
Of the organisins tested, B^ breve appeared to be the most sensitive to
chlorine treatment. The combined characteristics of sensitivity to adverse
- 51 -
conditions and ability to survive for only a relatively short period of time
outside the body sujport the proposal that Bifidobacterium species would be
useful irKlLcators of fresh fecal contamination.
B. lliB Isolaticfi and Identificaticn of Bifidobacteria frcm Fecal and Sewage
Samples by In-ja Huh (see Afpendix)
An attempt was made in this study to find an optimal medium for the
recovery of bifidobacteria from human and animal feces and from sewage. A
ccffrparison was also made with the HBSA medium designed to isolate sorbitol-
fermenting species of bifidobacteria. (In HBSA, lactose is replaced by
sorbitol) . I
Ihe results showed that the YN-17 medium yielded fewer false positive
target colonies than HBSA (5% and 10%, respectively) • Ihe majority of false
positive targets were found to be fecal streptococci. Lithium chloride, in
concentrations of 0.3% and 0.4%, was added to YN-17 and HBSA to inhibit
streptococcal grcwth; however, it was not successful in reducing the
contaminant levels in these media.
The YN-17 medium was developed by Mara and Oragui in 1983. It was found
in this study that colonies the authors described as bifidobacteria (i.e. blue
centre with pale green pjeriphery) were, in fact, a mixture of bifidobacteria
and streptococci. The characterization profiles indicated that the smaller
dark green/blue flatter colonies were bifidobacteria and those exhibiting a
pale green periphery were contaminating streptococci or, more frequently, a
mixture of the two organisms. Any colonies that were pale green in colour and
did not contain a darker centre were found to be streptococci.
Sorbitol-fermenting (B^. breve) species were isolated from human and cat
feces on the HBSA medium. It should be noted hcwever that none of the cat
isolates were able to ferment sorbitol in subsequent tube tests.
- 52 -
Bi f idobacterium adolescentis was the most cxanmon species of bifidobacteria in
sewage and human fecal samples. This organism was not found in cats.
The data shewed that compared with E^ coli levels, bifidobacteria counts
were 100 times greater in feces and 10 times greater in se^rage saitples.
C. A Study of the Survival of Bifidobacteria and their Rale in Vfater Quality
CCntrol by Sheila Shibata (see Appendix) .
The main objectives of this research project were to determine the most
suitable mediim for the isolation of bifidobacteria from feces and from
environmental samples and to compare the in vitro survival characteristics of
bifidobacteria and E^ coli. The results of the assessment of YN-17, MRS and
MFN media showed that the MRS medium recovered not only Bifidobacterium sp. but
high levels of lactobacilli and fecal streptococci as well. YN-17 and MEM were
equally selective for bifidobacteria but YN-17 had an advantage in that it
required a 24 h shorter incubation period. Bifidobacteria were found to exist
in feces at a 1000-fold higher concentration than E^ coli. The sorbitol-
fermenting bifidobacteria species, however, were present in feces in numbers
conparable with E^ coli (i.e. 10^) .
When bifidobacteria and E^ coli were added to dialysis diffusion chambers
and suspended in Lake Ontario water it was found that Bifidobacterium sp. died
off within 24 h of exposure vAiereas E^. coli died off more slowly and could
still be isolated after 48 h or more.
Since bifidobacteria are found in high levels in feces and have a tendency
to die off rapidly in the environment these organisms shew promise as
indicators of recent human fecal pollution.
- 53 -
D. Clostrldimn perfrinqens and Bifidobac±erii3m sp. as Trac3ers in Storm Sewers
by Eric Hani (see /^peixiix)
This project examined the feasibility of using C^ perfrinqens and
Bifidobacterium sp. as indicators of fecal pollution from warm-blooded animals.
The data shewed that both indicators were present in high concentrations in
feces but that bifidobacteria could be isolated on a more consistent basis.
The graphs indicated that bifidobacteria levels were hi(^ in both sanitary and
hi^ priority storm sewage. Clostridium perfrinqens densities, on the other
hand, were conparatively lew in the hi^ priority storm sewage. The histogram
giving the geometric mean concentrations of E^ coli, C^ perfrinqens and
bifidobacteria showed that levels of all three indicators tended to be hi(^er
in the sanitary and high priority storm sewage sairples in conparison with the
non-priority storm sewer sairples. Clostridium perfrinqens could not be
recovered fran one non-priority storm sewer sanple. There did not appear to be
any major trends with respect to source vhen the ratios of the three indicator
orrganisms were coirpared (Table E-1) .
In a random sanpling of human, cat and dog feces, bifidobacteria levels
were found to be hi^er than E^ coli in humans. Although C^ perfrinqens was
recovered frcm the cat fecal saitple, no bifidobacteria were found. As
previously mentioned, Mara and Oragui (1983) found sorbitol fermenting species
of Bifidobacterium to be exclusive to human feces. However, this reaction does
not appear to be a practical criterion for source tracing since only a small
percentage of the sanitary sewage isolates were sorbitol fermenters.
- 54 -
E. Chctracterizaticns of Pseudciiicr>as aerucrinosa fran Storm and Sanitary Sewers
by Rita Harmandayan (see i^pendix)
This project was designed to investigate the use of serotyping and
genotyping to distinguish P^ aeruginosa strains fron human and non-human
sources. The data shewed that levels of P^ aeruginosa were close to 1 x lO'* in
sanitary sewage and 1 x lO-'- in storm sewage sanples. Approximately equal
numbers of isolates fron both sources were serotyped using the Difco serotyping
kit number 3081-32 (Pseudomonas aeruginosa antigen kit). Serotype 0:6 was the
most frequently indentified serotype in both the storm and sanitary sewers.
Serotypes 0:1 and 0:11 were also commonly isolated from both sources. Serotype.
0:16 was recovered from storm sewers only, with a frequency of 5.4%. Serotype
0:10, on the other hand, was isolated solely from sanitary sewers with a
frequency of 21.9%. It is possible, therefore, that the presence of type 0:10
may be indicative of human fecal contamination.
To determine if identical genotype patterns were present within each
serogroup, restriction enzyme analysis was performed on isolates with common
serotypes. Of the six different restriction enzymes used to cut the genomic
K^A, Sma I produced the best banding pattern on polyacrylamide gels. The
results shewed that not all isolates with the same serotype possessed the same
genome pattern. Ihis is e>plained by the fact that the genes responsible for
serotyping the 0-Ag comprise a small percentage of the total genome and that
other genes besides those that code for the 0-Ag will appear v*ien the total
chromosomal CNA is cesrpared. Ihus, the REA technique was shown in this study
to be highly specific; it further subdivides one serotype into different
genotypes. In future, a probe could be developed from either total chromosomal
OIA or a specific fragment common to human strains that would allow
hybridization against other P^ aeruginosa strains. The probe would help to
differentiate between isolates from human and non-human fecal waste.
- 55 -
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■ - 58 -
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Infec. Dis. 130 (suppl.): S47-S52.
- 60 -
APPENDIX A
Tsitle 14
Sanpling Dates far Dry and Wet Weather Surveys
Year
Date
category
1986
1987
1988
October 21, 22 and 28, November 18
June 10, 11, 12 and 19
August 17
June 20, 21, 28
August 15, 22
Dry Weather I
Dry Weather II
Dry Weather
Dry Weather
Dry Weather
1987
July 14
September 18
October 21
Wet Weather I
Wet Weather
Wet Weather
- 61 -
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- 116 -
AFraNDIX B
CCMPARATIVE STUDY OF THE SURVIVAL
OF INDICATOR BACTERIAL SPECIES
Eric Bauer, Department of
Microbiology, University of Toronto
- 117 -
TABLE OF OCWTEyiS
Page No.
I. INTRDDUCnON 121
II. OBJECnVES OF RESEARCH 126
III. MATERIALS AND METHODS 127
Bacterial cultures 127
Growth cycle determination 127
Membrane filtration techniques for the isolation 129
of test organisms
Enumeration of colony forming units per mL of 130
test bacterial cultures
Chlorine test procedure 131
IV. RESULTS
Growth cycle determination 134
Bactericidal effect of chlorine 138
V. DISCUSSION 155
VI. RECCMIENDATIONS • 162
VII. APPENDIX 163
VIII. REFERENCES 169
• - 118 -
UST OF TARTRR
Table
Page
B-1 Media and Incubation Parameters for Enumeration 128
of Selected Bacterial Groi-ps
B-2 Colony Forming Units per Millilitre at Rocan 135
Terrperature
B-3 Colony Forming Units per Millilitre at 15 136
degrees Celsius , • ,
B-4 Effect of Chlorine at a pH of 6.0 ^ ' 150
B-5 Effect of Chlorine at a pH of 9.0 150
B-6 Effect of pH on Forms of Chlorine 160
- 119 -
UST OF FIGURES
Figure Page
B-1 Colony Forming Units per mL vs. Time at Room 139
Temperature
B-2 Colony Forming Units per mL vs. Time at 15 140
degrees Celsius
B-3 E. coli CFU/mL vs. Time at Room Tenperature 141
and 15 degrees Celsius
B-4 P. aeruginosa CFU/mL vs. Time at Room 142
Temperature and 15 degrees Celsius
B-5 Strep, on m-Ent CFU/mL vs. Time at Room 143
Tenperature and 15 degrees Celsius
B-6 Strep, on m-E CFU/mL vs. Time at Room 144
Temperature and 15 degrees Celsius
B-7 B. longum CFU/mL vs. Time at Room Terrperature 145
and 15 degrees Celsius
B-8 Magnification of Colony Forming Units per mL 146
vs. Time at Room Tenperature
B-9 Magnification of Colony Forming Units per mL 147
vs. Time at 15 degrees Celsius
B-10 Magnification of E. coli CFU/mL vs. Time at 148
Room Tenperature and 15 degrees Celsius
B-11 Magnification of P. aeruginosa CFU/mL vs. Time 148
at Room Temperature and 15 degrees Celsius
B-12 Effect of 0.2 ppm Chlorine on Various Organisms 151
at a pH of 6.0
B-13 Effect of 0.4 ppm Chlorine on Various Organisms 152
at a pH of 6.0
B-14 Effect of 0.2 ppm Chlorine on Various Organisms 153
at a pH of 9.0
B-15 Effect of 0.4 ppm Chlorine on Various Organisms 154
at a pH of 9.0
- 120 -
UST OF ABEREVIATrONS
Organisms :
E. coli - Escheric±iia coli
B. breve - Bifidobacterium breve
B. lonqum - Bi f idobacter ium longum
P. aeruginosa - Pseudomonas aeruginosa
S. faecal is var, faecal is - Streptococcus faecal is
variety faecal is
S. faecium var. casseliflavus - Streptococcus faecium
variety casseliflavus
CFU - Colony forming unit
°C - Degrees celsiiis
dH20 - Distilled water
E - Exponential
pH - Hydrogen ion concentration
mL - millilitre
ppm - Parts per million
PSI - Pounds per square inch
vs. - versus
M - nxDlar
- 121 -
nraojJCTicN
Fundamental to the interpretation of data concerning surface
uater quality is an understanding of the growth cycles and viability
of fecal indicator bacteria and enteric pathogens disseminated by
waterways. In the recent past public awareness pertaining to the
contamination of surface waters has resulted in increasing efforts
to control this type of pollution. The potential health hazard, in
v/hich waterways may act as both a vehicle and a reservoir for
agents of infectious disease, is apparent. The prevention of.
epidemics caused by such organisms as Salmonella typhi. Salmonella
typhimurium. Vibrio cholerae . Shigella species, Giardia lamblia,
enterotoxigenic E. coli and P. aeruginosa may only take place with
the adequate control of fecal contamination (Gyles, 1984) .
The main concern of this study deals with the comparative
analysis of the life cycle of E. coli, P. aeruginosa , S. faecal is
var. faecal is, S. faecium and B. longum. The theoretical aspect
of this investigation is concerned with obtaining an organism of
short life duration so that it may be used as an indicator of
recent fecal contamination. Although the detection of fresh fecal
contaminants within any surface water body is paramount, the
impetus for this research came from the ever growing problem of
storm water sewage contamination. It is for this reason that
growth curves for the indicator organisms were established at both
room tenperature and 15°C (approximate sewer tenperature) .
■ - 122 -
The original design strategy behind storm water sewage
channels was the alleviation of excessive runoff - water from
urbanized areas. Theoretically these storm sewers should have no
human fecal input, hcwever, as reported by the Toronto Area
Watershed Management (TAWM) , this is not the case. Fecal
pollution within the Humber and Don Rivers does result in storm
water sewage serving as a contributor.
The organisms selected for this study were chosen for the
following reasons:
E. coli ' '
This organism is constantly found in the human intestine
in large numbers.
The fate of the coliform bacteria reasonably reflects
that of the pathogenic bacteria, althou^ the life
e>pectancy of the fecal coliforms is normally longer
than that of intestinal pathogens.
This organism is easy to isolate and enumerate in the
laboratory environment and is normally not pathogenic
(Schuettpelz, 1969).
S. faecal is var. faecal is / S. faecium
A hi^ percentage of the strains have properties which
enable their source to be identified with considerable
certainty.
Members of the fecal streptococci group are often used
to signify the presence of intestinal pathogens
(McFeters et al., 1974).
- 123 -
P. aeruginosa
Ringen and Drake (1952) have shewn that this organism
was not isolated from locations free of human habitation
or waste material.
This opportunistic pathogen is particularly associated
with the hi^ incidence of otitis externa in surface
water related areas (Levin and Cabelli, 1972) .
B. lonqum
The strictly anaerobic requirement of this organism
makes its inclusion in a study of growth within a sewer
environment of interest.
A relatively small amount of work has been done on this
organism and its membership in the group of fecal
organisms.
The method of choice for the enumeration of the indicator
organisms was that of membrane filtration. This technique was
selected because of the relative sinplicity of the procedure, the
capability of obtaining results within 24 hours, instead of the 48
to 96 hours demanded by the Most Probable Number test and
finally, the larger volume of samples v*iich may be analyzed ma3djTg
the obtained results more representative.
The second portion of this survival research project
concerns iself with the effect of chlorination on the following
organisms: E. coli. P. aeruginosa. S. faecium var. casseliflavus
and B. breve. The lethality of uncombined chlorine, in the form
of unionized hypochlorous acid has made this chemical the most
- 124 -
widely used reagent for the disinfection of water distribution
systems and reservoirs (Ridgway and Olson, 1982) . Of more recent
interest, hcwever, has been the possible application of the method
of chlorination for purposes of sterilization. Factors such as
concentration of the chlorine used, the pH of the enviromnent in
vv*iich the chlorine is dispensed and the free chlorine residual
must all be considered vdien analysing the effectiveness of this
agent as a bactericide (Seyfried and Fraser, 1979) .
The action of chlorine, from the chemical point of view, may
be summarized as follows: '
1. Hydrolysis of chlorine:
CI2 + HOH =- HOCl + ir^ + CI"
Above pH values of 5.0, the hypochlorous acid
(HOCl) dissociates in aqueous solution and forms an
equilibrium with hydrogen and hypochlorite ions,
with the relative amount of each species dependent
upon the pH.
2. Dissociation of hypochlorous acid:
HOCl = H^ + OCl"
The bactericidal activity is a result of the
hypochlorous acid \«*dch is free chlorine, while the
hypochlorite ion has limited killing ability, but
acts as a reservoir of available chlorine.
3. Combined or "stabilized" chlorine:
- 125 -
Ihis is the reaction of chlorine with such
materials as ammonia or its salts, cyanuric acid,
sulfamic acid or urea. Chlorine in this form is
virtually ineffective, having a killing time of
approximately one thirtieth that of hypochlorous
acid (Black et al., 1970).
The final point of interest concerning the action of
chlorine is related to its biochemical action upon the bacterial
cell. It is at this point that differences in lethality exist,
when environmental conditions are kept constant between organisms.
Recent proposals, by Canper and McFeters (1979) as to the mode of
action of chlorine on the bacterial cell include:
unbalanced metabolism after the destruction of key
enzymes
disruption of protein synthesis
oxidative decarboxylation of amino acids
reactions with nucleic acids, purines and pyrimidines
formation of chloro derivatives of cytosine
creation of chromosomal abberations
induction of deoxyribonucleic acid lesions with the
acconpanying loss of deoxyribonucleic acid transforming
ability
inhibition of oxygen uptake and oxidative
phosphorylation coupled with the leakage of some
macromolecules
- 126 -
OBJECTIVE OF RESEARCH
The objectives of this research project were to:
1. Study the growth cycle of E. coli. P. aeruginosa, £5.
faecal is var. faecal is, S. faecium and B. longum. The
results of this corrparative analysis may then be applied to
the development of a methodology for detecting the source of
sanitary waste pollution in surface water bodies, since the
presence of a short life expectancy organism will be
indicative of recent pollution.
2. Of growing interest is the use of chlorine, not only as a
disinfectant, but also as a potential sterilant.
Manipulation of the concentration of chlorine, as well as
the hydrogen ion concentration of the environmental setting,
will provide insight into the effective lethality of chlorine
on E. coli, P. aeruginosa , S. faecium var. casselif lavus . and
B. breve.
- 127 -
MATERIALS AND METHODS
3.1 Bacterial cultures:
Seven bacterial cultures were used in this study.
Four laboratory strains v^iich included E. coli, P.
aeruginosa , S. faecal is var. faecal is, and S. faecium.
Two environmental isolates which included B. longum and
S. faecium var. casseliflavus.
One fecal saitple isolate, B. breve, taken from a
chicken.
The environmental isolates and the fecal sample isolate were
positively identified using biochemical testing procedures, as
outlined in Bergey's Manual of Determinative Microbiology (1985) .
3.2 Growth cycle determination:
E. coli, P. aeruginosa , S. faecal is var. faecal is and S.
faecium were all grcwn for 24 hrs. , aercbically, in lOmL
of nutrient broth, at 35°C.
B. lonqum was grown for 48 hrs., anaerobically, in lOmL
of MRS broth at 35°C.
Two 250mL culture flasks were autoclaved for 15 minutes
at 121°C / 15 PSI. To these flasks, 90mL of filter
sterilized pond water was then aseptically added.
To ensure that sterility was achieved, spread plates of
the sterilized water were made on nutrient agar.
Once these culture flasks were prepared, the five
cultures
- 128 -
Table B-1
Media and Incubaticn I^rameters for
Qiimpratlcn of Selected Bact-prial Grtx^s
BACTERIAL
0«XJP
ZNCUBAT- TIME
MEDIUM* TEMP 'C (HRS)
TARGET Cr)I£NY ICREHDLDGY
E. Coli
m-TEC 44.5 23+1 - flat greenish/yellow colonies
- circular
NA
35
24 - small beige colonies
- circular
P. aeruginosa
m-PA 41.5
48
convex brownish-green or tan
circular
NA
35
24
spread out and of irregular
form
S. faecal is
var. faecal is
m-EOT
35
48
maroon, red, or pink colonies
circular and raised
S . f aecium
m-E
41.5
48
blue/pink colonies
pulvinate to umbinate
S^ f aecium BHI
var. casseliflavus
35
24
very small yellow colonies
convex to pulvinate
B. loncami
YN17
Blue
35
48
smooth to undulating surface
convex to pulvinate
soft, moist, slimy blue
colonies
B. breve
MRS
35
24
convex to pulvinate
ivory coloured
muccoid and soft
- 129 -
were each diluted in lOmL of their respective broths and then aseptically added to
the culture flasks.
These dilutions were pr^)ared so as to start with an initial colony
forming units per mL of approximately 10E5 to 10E6.
The bacterial culture dilultions were made in duplicate, as were the
culture flasJcs, in order that the growth cycles could be examined at both
15°C and room tenperature.
Both the tenperature of the 15°C incubator, as well as the tenperature of
the laboratory were recorded daily:
Room tenperature range - 23°C +/~ 2°C
15°C incubator - 15°C +/" 2°C, with the exception of Day 9
of the study when the incubator tenperature
fell to 8°C.
3.3 Membrane filtration technique for the isolation of the test organisms.
The procedure followed for the membrane filtration was that as outlined in
(Standard Methods for the Examination of Water and Waste Water, 1971) .
Sanples were removed from both the 15°C and the room tenperature culture
flasks over a 40 day experimental period.
The first determination (Day 0) was taken immediately following the
initial incubation of the bacterial cultures within the pond water.
- Sanple filtration, for the purpose of enumeration, was always carried out
between 9:00AM and 12:00FM and the determination dates were recorded.
Sanples were first diluted in 99mL phosphate buffer dilution blanks (see
i^pendix) and then filtered (Gelman filters) .
^^proximately lOOmL of phosphate buffer wash water (see Appendix) were
- 130 -
divided into three aliquots for consecutive rinsing of the saitple and
membrane filtration apparatus.
- After washing the membrane filters were placed on the growth medium,
taking care to ensure that no air bubbles were trapped under the filter.
The growth medium, incubation parameters and target morphology for all of
the test organisms may be found in Table 1.
3.4 Enumeration of colony forming units per mL of test bacterial cultures:
- For all organisms, with the exception of B. lonoum, target colony
morphology was specific to the isolating growth medium.
The ability of fecal streptococci to grow on the YN17 Blue
(Bifidobacterium isolating) medium made morphological examination of
typical colonies from this medium essential.
- Typical colonies were selected frcm the membrane filter, from the YN17
Blue medium and microscope slide preparations were made.
- Bi f idobacter ium characteristic morphology; long curved, club-shaped,
swollen or dumb-bell shaped rods, vs*iich may also be bifurcated.
- At the point in time when this type of cell morphology was no longer seen,
typical colonies were selected and exposed to biochemical testing
(Bergey's Manual of Ceterminative Microbiology, 1985).
Ihe two fecal streptococci, S. faecal is var. faecal is and S. faecium, were
both included into the study so that it could be determined which had a
shorter life span. Typical colonies were selected from the m-E medium
(see Appendix) and biochemically tested so as to permit the calculation of
a ratio of surviving numbers of the two fecal streptococci.
- The fecal streptococci were also grcwn on both m-Ent and m-E (see
- 131 -
Appendix) so as to cxmpare the consistency of counts between the colony formir.--
units on the two different media.
The appearance of the colonies on m-E agar was variable, and biochemical
tests were perfornved on two different types of colonies in order to detenr.ir.o
whether these differences represented the two different fecal streptococci .
3.5 Chlorine test procedure:
The bacterial effect of chlorine on the test organisms was determin&.l
using the experimental procedure of Seyfried and Fraser (1980) .
All glassware which came into contact with the experimental chlorir.-:-
concentrations was acid-washed and then treated overnight with a calcicc.
hypochlorite solution of concentration O.Olg per lOOOmL.
A stock chlorine solution of 0.5g calcium hypochlorite to 500mL of dii.L
was prepared. A sterile sodium thiosulfate solution (0.35g / 500mL) -..a?-
also prepared and was used to neutralize the chlorine.
Reaction tubes were filled with 8mL of dH20 and then autoclaved for i-I
minutes at 121°C / 15 PSI. With sterilized IM sodium hydroxide and :
hydrochloric acid the pH of the reaction vessels was then adjusted to
and 6.0, respectively.
E. coli. P. aeruginosa and S. faecium var. casseliflavus were all grov.Ti
for 24 hours, aerobically, in lOmL of nutrient broth at 35°C.
B. breve was grown anaerobically for 24 hours in lOmL of MRS broth at 35°C.
Two variables were incorporated into the experimentation of the
- 132 -
bactericidal effect of chlorine on the test organisms, through the follov.-i:-.o
experimental trials:
(A) Effect of chlorine at a concentration of 0.2 ppm and a pH of 6.0
(B) Effect of chlorine at a concentration of 0.2 ppm and a pH of 9.0
(C) Effect of chlorine at a concentration of 0.4 ppm and a pH of 6.0
(D) Effect of chlorine at a concentration of 0.4 ppm and a pH of 9.0
Bacterial cultures were centrifuged and then washed twice with 5mL aliqucts
of sterile dH20. The washed cultures were then resuspended in 5mL c:
sterile dH20 and thoroughly mixed.
The estimation of the chlorine concentration was done before each experimenCLi.
trial and made use of the diethyl-p-phenylenediamine (DPD) test (Americar.
Public Health Association, 1971) .
At this point l.OmL of culture was removed and diluted in 99mL of phospharc-
buffer dilution blank. This suspension was then placed on ice for lat^-r
further dilution and subsequent membrane filtration. In this manner •::._
initial concentration of bacteria per mL could be calculated.
The test system consisted of 8mL of sterile pH adjusted dH20, ImL of test
culture suspension and ImL of chlorine.
The cells were exposed to the particular chlorine concentration for i
minute. After this contact period ImL of the neutralizing solution of :
sodium thiosulfate was added to stop the reaction.
ImL of suspension was then removed from the reaction tube and placea ...
the 99mL phosphate buffer dilution blanks.
Membrane filtration was performed first on the chlorine exposed cells ar.L;
then on the initial cells which were on ice until this point. Each teiic
- 133 -
organism was filtered individually, at the "before and after" e>posures,
so as to reduce the possibility of any cross contamination.
Referring to Table 1, the grcwth media, incubation parameters and typical
colony morphology may be seen (Note: In this case selective media was not
used. Instead, less stressful media was chosen; nutrient agar, BHI agar
and MRS agar) .
Ihe experimental procedure was performed at a teitperature of 23 +/~ 2°C.
• - 134 -
RESULTS
4.1 Growth cycle determination:
Tables 2 and 3 shew the growth of E. coli, P. aeruginosa. S. faecal is var.
faecal is / S, faecium on m-Ent and m-E media, and B. lonqum. Enumeration of test
bacterial cultures was performed at room tenperature and 15°C (Table 2 and Table 3,
respectively) .
As can be seen in the data of the rocm tenperature detennination, the fecal
streptococci attained the highest numbers of colony forming units per mL. Although
all organisms showed a period of substantial increase in growth, this period was
extremely short for B. lonqum, lasting for only the first day. In conjunction with
this, B. loncpjm was the first organism to die off; as well, P. aeruginosa was the
only other organism to die before the end of the 40 day experimentation period.
Some fluctuation in growth was recorded, namely the data collected does not reflect
a steady increase in growth follcwed by a decline period. Examination of the
results of the fecal streptococci illxostrate a high degree of consistency between
the colony forming unit per mL values on m-Ent with that of m-E.
At the 15°C determination, the most striking feature is the rapid "die-off" of
E. coli, P. aeruginosa and B. longum. These three organisms all perished sometime
between Day 8 and Day 13. As with the rocm tenperature determination, the fecal
streptococci again were most abundant; however, the numbers obtained at 15°C were
not quite as high as those at room tenperature. Another notable occurrence is that,
- 135 -
Table B-2
Colony Forming Units per Mlllllitre at Room Temperature
TIME
E. coll
aaaaasaaaaasa
P. aeruginosa
Strep,
on Ment
isaaaaaaaaaaa:
Strep,
on ME
ssaaaaaaassssa:
i.'
ssaas
, lonqum
(days)
saaaaaaa:
0
1.80OB+05
2.695E-t-05
1.320E-f06
1.815E+06
5.
.840E-I-05
1
2.330B+07
1.090E^06
3.980E+06
3.730Ef06
7.
.610E+05
4
3.750E+07
1.875E+06
4.390E+07
5.540E+07
5,
,370E+05
5
3.070E+07
3.480Ef06
1.223E+08
1.383E+-08
5,
.025E+05
6
1.925E+07
1.800E-I-06
1.335E+08
1.250E+O8
1,
.395E+05
7
4.900E+07
1.123Ef06
6.333B+07
1.405B+08
2.
.230E+05
11
4.120E+07
2.880E+07
5.750E+07
1.363E4-08
9,
.720E+04
13
3.950E+07
4.0O0E+07
2.330B+09
6.150E+07
0.
.0OOE4-OO
15
4.500B+07
7.850E-t-08
3.790E+08
1.920E+09
0.
.OOOE+00
19
2.100E+07
8.640B+08
3.180E-I-08
5.960E+08
0,
.OOOE+00
21
1.840E-«-07
3.500E+07
2.880E+07
1.660E-»-08
0.
.OOOE+00
25
1.760E+07
4.740E^06
2.610B+07
3.910E+07
0.
,0OOE+OO
27
1.400E+07
6.320E+05
2.800E+07
4.500E+07
0,
,OO0E+OO
29
9.200E+06
4.640E4-04
2.700E+07
3.900E+07
0,
.OOOE+00
36
6.800E-t-06
1.300E+02
2.400E+07
3.000E+07
0.
.OOOE+00
40
8.340E+05
0.000B4-00
1.240E+04
1.840E+07
0,
, OOOE + 00
- 136 -
Table B-3
Colony Forming Units per Mlllllltre at 15 degrees Celsius
TIME
E. coll
P. aeruqlnosa
Strep.
Strep.
!-•
. lonqum
(days)
on Ment
on ME
asasass
asssaaassasa:
saaaaaaasaaaaaass
:aasasaaaaaaa
aaaaaaaaaaaaaaaa
aaaaaasaaa:
0
2.020E+05
3.170B+05
1.350B+06
1.625E-I-06
4 •
,900E-t-05
1
1.420E-«-05
1.903E-^05
1.453E-t-06
1.500E-f06
4 .
.450E-t-05
4
1.260B+05
2.760E-»-05
4.640E^06
4.685E+06
4
.210E+05
5
8.850E+04
1.085E-t-05
9.850E-t-06
1.005E-I-07
1 •
.873E-h05
6
2.020B+05
3.245E4-05
4.800E-t-07
3.070Ef07
9
.730E+04
7
2.087E+06
3.200E-«-04
3.270E-t-08
2.880E-t-08
8
.060E-I-04
11
4.100E-I-04
O.OOOE-t-00
1.900E-t-08
2.437Ef08
2
.120E+04
13
2.000E+02
O.OOOE-t-00
2.720E-t-08
2.725E-t-08
0,
.OOOE-i-00
15
O.OOOB+00
O.OOOB+00
2.310E+08
2.040B>08
0.
.000E4-00
19
O.OOOE+00
O.OOOE-t-00
1.130E-t-08
1.090E-I-07
0,
.OOOE-l-00
21
O.OOOE+00
O.OOOE+00
7.650E-I-06
9.130B-I-06
0,
,000E+00
25
O.OOOE-l-00
O.OOOE-t-00
1.190E-t-04
1.850E-I-04
0,
.OOOE-l-00
27
O.OOOE+00
O.OOOE-t-00
2.400E^04
2.200E-I-04
0.
,000E+00
29
O.OOOE-t-00
O.OOOE-i-00
1.900E-I-04
2.500E-t-04
0.
.OOOE-fOO
36
O.OOOE^OO
0.000E4-00
1.400E^04
2.800E<-04
0.
.OOOE+00
40
O.OOOE-t-00
O.OOOE-t-00
1.240E-t-04
2.330E-I-04
0.
,OOOE-l-00
- 137 -
with the exception of the fecal streptococci on m-Ent, all other cases showed a
decline immediately after incubation was initiated. Again, a degree of fluctuation
within the growth cycle of the organisms was observed upon enumeration.
In the case of B. longum. at both room temperature and 15°C, microscopic slide
preparations of colonies from the Day 11 determination did possess cells of
characteristic Bi f idobacterium morphology. On the Day 13 enijmeration, however,
morphological analysis proved negative for Bifidobacteriiim and for this reason four
typical colonies were selected from a YN17 plate containing 5 colonies, for
biochemical testing (Bergey's Manual of Determinative Microbiology, 1985). The
results obtained were all negative for Bi f idobacterium . but positive for
Streptococcus . Due to the absence of positively identifiable Bifidobacterium all
colonies v\^ch did grew were not enumerated and it was assumed that B. longum no
longer existed.
Figures 1 through 10 represent the graphical analysis of the results recorded
in Tables 1 and 2. The following is an explanation of v*iat the respective Figures
depict :
Figures 1 and 2 are comparative graphs of the test organisms at both room
temperature and 15°C. (Note: These Figures, as with all others in v*iich the fecal
streptococci are found, do not distinguisli between S. faecal is var. faecal is and S.
faecium but instead conpare the combined fecal streptococci as to their growth on
either m-Ent or m-E growth media) . As can be seen on these graphs, the values for
the fecal streptococci are enormous in conparison to the other organisms. For this
reason, magnifications of these grap^is were required and may be found in Figures 8
and 9. The fluctuations in growth are more readily appcirent when viewing the
graphs, but in an attempt to compensate for those days on v*iich determinations were
not taken, the "best-fit" line was drawn through the points. Two final points
- 138 -
worthy of mention include the rapid "die-off" of all onganisms but the fecal
streptococci, at 15°C. Secondly, the fact that only E. coli demonstrates a period
of growth followed by a short period of "die-off" and then another resurgence, at
both tenperature settings.
Figures 3 through 7 plot the results obtained at room temperature against those
of 15°C, for each individual organism. Ihis permits the viewing and conparison of
the tenperature settings, on the grcwth of the test organisms, without the added
confusion of the results from the other organisms. In all cases the numbers of
colony forming units per mL are drastically reduced at 15°C. This difference was so
marked with respect to E. coli and P. aeruginosa that magnifications of their
results were produced and may be found in Figures 10 aind 11. The fecal
streptococci and B. lonqum seemed to suffer less from the reduced tenperature of
15°C, rarely having CFU per mL enumerations which differed by even an order of
magnitude.
4.2 Bactericidal effects of chlorine:
Tables 4 and 5 contain the results of chlorine exposure upon the test organisms
E. coli. P. aeruginosa. S. faecium var. casseliflavus and B. breve. For all
experimental situations, the exposure time to chlorine was for a 1 minute contact
period. Table 4 is a record of the results obtained v*ien the pH of the reaction
flask was 6.0, vtiile Table 5 illustrates the results obtained at ftfi 9.0.
The fact that the bactericidal effect of chlorine is not only exposure and
concentration dependent becomes quickly apparent when the percentage of organisms
killed is conpared between the different pH settings. Also, the higher concentration
of chlorine is much more lethal, at both pH. settings, than is the 0.2 ppm
concentration.
- 139 -
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- 149 -
One ofcservation of particular interst is the tendency towards more effective
killing, by chlorine, when it is functioning within an acidic environment. Only E.
coli and S. faecium var. casseliflavus had a Icwer killing percentage (at 0.4 ppm)
at the Icwer pH of 6.0. All other experimental determinations shewed more effective
killing at pH 6.0. Ihis observation was particularly apparent in the killing of P.
aeruginosa. ' ^ ^
Figures 12 tlirou^ 15 graphically d^ict analysis of the results of the
chlorine experimentation. It is obvious that chlorine has lethal effects, from the
negative slope of all the graphs, but more inportant is the drastic effect seen at
0.4 ppm in coirparison to that of 0.2 ppm. At the two ends of the percentage killed
spectrum lie E. coli. with the smallest percentage killed (0.2 ppm at pH at 9.0) and
B. breve , experiencing the highest percentage killed (0.4 ppm at pH 6.0) .
- 150 -
Table B-4
Bffect of Chlorine at a pH of 6.0
ORGANISM INITIAL CFU/nL
E. coll
^. aeruginosa
3 . faeclun
1.99B+09
2.38E+09
1.69B4-09
var . cassellf lavus
B. breve 2.67E+09
AFTER CHLORINE ADDITION
0 . 2 ppa 0 . 4 ppa
3.1584-08
1.77E+08
1.52B4-08
2.82E-f08
1.31B-t-08
8.25E+07
8.65B4-07
5.40E+07
\ KILLED
0 . 2 ppn 0 . 4 ppm
84.2 93.4
92.6 96.5
91 94.9
89.4
98
Table B-5
Bffect of Chlorine at a pH of 9.0
ORGANISM
E. coll
£^. aeruginosa
^. faecltim
ar. cassellf lavus
INITIAL CPU/mL
AFTER CHLORINE ADDITION
0 . 2 ppm 0 . 4 ppm
3.80E-»^09
1.82B4-09
2.80E-I-09
1.08E-I-09
3.47B4-08
2.73E+08
2.30E-t-08
1.90B4-08
1.20B-(-08
\ KILLED
0.2 ppm 0 . 4 ppm
71.6 93.9
80.9 89.6
90.3 95.7
B- breve
6.15S-I-09
1.04E4-09
3.16B-»-08
83.1
94.9
- 151
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DlSOJSSICa^
The analysis of the growth cycles of E. coli, P. aeruginosa, S. faecal is var.
faecal is, S. faecium and B. loncrum had as its goal the development of a methodology
for the purpose of locating a bacterial indicator vrtiich could be used for the
detection of recent fecal contamination within surface water bodies. This is a
problem of growing public concern, since the potential health hazard such pollution
creates is of grave inportance. The experimental trial at room teitperature served
to act as a standard or means of ccmparison for those results obtained at 15°C.
This teitperature was selected to simulate, on the basis of tenperature alone, the
environment of a storm water sewer (Seyfried personal communication, 1988) . It has
been reported by the Toronto Area Watershed Management (TAWM) that a major
contributor to fecal contamination within the Humber and Don Rivers is the effluent
from Toronto storm water sewers.
The controversy over v*iich bacterial indicator to use, for the purpose of water
pollution control policies, has resulted in much debate. Taylor et al. (1973),
state that the enumeration of fecal coliforms provides a more precise measure of the
potential health hazard in surface water bodies, than does the more omniscient total
coliforms. According to Doran and Linn (1979) fecal coliforms are reported to be
the most reliable indicator of fecal pollution of water; however, with these
organisms the source of contamination may not be identified. To compensate for this
the use of fecal streptococci has been suggested in order to differentiate fecal
contaminants from human or other animal sources (Geldrich and Kenner, 1969) .
Schuettpelz (1969) reports that enterococci approach numbers of total coliforms in
sewage, but have the added advantage of not multiplying in water.
It would appear that the trend to employ fecal streptococci as indicators of
fecal pollution is growing, but problems with their acceptance still exist.
- 156 -
Firstly, fecal streptococci are present in smaller numbers in feces, sewage and
polluted waters, and the easier quantification of fecal coliforms is inhibitory to
their use. Ihe second major problem lies within the confusion which exists
concerning the identity of fecal str^Jtococci, in particular, that of their
ecological distribution (Levin et al. 1975) . To overcame this handicap a barrage of
research has been performed in the field of developing media selective for specific
fecal streptococci, so that their identity may be positively confirmed. Work done
by Isenberg et al. (1970), Wie-Shing Lee (1972), Switzer and Evans (1974) and
Brodsky and Schiemann (1976) is illustrative of only a minor saitpling of involved
participants. Another attempt at overccining the barrier of being unable to
identify the source of the fecal contamination has been to use the fecal coliform
(FC) to fecal str^atococci (FS) ratio. Doran and Linn (1979) claim that an FC/FS
ratio of greater than four is usually indicative of domestic waste water pollution,
v*iile an FC/FS ratio of less than 0.7 may be associated with non-human animal
wastes.
Recently, P. aeruginosa has been considered as a potential indicator of fecal
contamination from human sources. Wheater and co-workers (1978) have shown that P.
aeruginosa was not found in a variety of animal feces and also that the presence of
this organism in animals and soil was due to chance and the close proximity to man
(approximately 15% of the human population contain P. aeruginosa as a norrral
commensal microbe) , For this reason and because of the opportunistic pathogenic
nature of P. aeruginosa it has been given consideration as an indicator of fecal
contamination in surface water bodies.
The final organism, B. longum, which was included in this growth study
comparison is a member of a genus upon which a great deal of research has not been
done. Classification and identification of Bifidobacterium species monopolizes the
- 157 -
research of this organism. Its membership as an intestinal bacteria and
siibsequently as a fecal organism protpted its inclusion.
Concerning the 15°C temperature setting, experimental results shewed that P.
aeruginosa had the shortest life span. As well, E. coli (fecal coliform) and B.
lonqum have life expectancies only slightly longer than that of P. aeruginosa. The
two fecal streptococci survived longest, and were still graving when experimentation
was terminated. The problem encountered here was that the continued growth of both
S. faecal is var. faecal is and S. faecium prevented the determination of which
oinganism died first. Biochemical testing done at Day 30 on the fecal streptococci
resulted in the identification of both species, with continued "picking" being
unfeasible because of time limitations.
Despite the stated goal of this reasearch, namely the determination of a
bacterial indicator of recent fecal contamination of surface water bodies, the
theoreticcil approach was to simulate an environment like that of a storm sewer,
since these conduits act as a major vehicle for the transport of fecal pollutants.
In this manner the source of the contamination is addressed, as opposed to
identifying and then atteirpting to treat the surface water bodies which have been
defiled. E. coli, P. aeruginosa and B. longum seem to all possess the criteria of
having a short life. Therefore, for pfurposes of identifying the newness of fecal
contaminants they could feasibly be employed. Hie experimental data collected
indicates this conclusion, however, environmental concern regarding this topic does
not end with sinply identifying how recent the pollution is, it also requires the
differentiation of human and animal fecal contamination. The human input into the
problem of surface water pollution may, with p^erseverance and regulation, be
controlled; however, for the animal contribution this task would not be practical.
Thus it becomes essential to discriniinate between human and animal fecal waste,
- 158 -
which as per the literature seems to necessitate the use of identifiable fecal
streptococci .
The second portion of this research study was involved with examining the
bactericidal effect of chlorine upon E. coli. P. aeruginosa. S. faecium var.
casseliflavus and B. breve. The wide acceptance of chlorine for purposes of
disinfection stimulated interest in vy*iether or not chlorine, under appropriate
conditions could be used as a sterilant. The fact that this research project began
as a survival study must also not be overlooked. The two parameters v^ich were
given consideration in this study included the pH of the environment in v^ich the
chlorine functioned, as well as the particular concentration of the chlorine
exposure dosage. The length of application of chlorine was not manipulated, being
kept constant at one minute for cill trials.
According to Caitper and McFeters (1979) e^qxDsure of waterbome organisms of
fecal origin to hostile chemical environments results in a chain of events. The
first of these is stress, which is then followed by injury and finally, death. If
microorganisms have been exposed to chlorine, as in this experimentation, then the
cultivation of survivors necessitates the use of a non-selective (not stressful)
growth medium. For this reason, the antibiotic free nutrient, EHI, and MRS agars
were used. In some cases it has been suggested that the injuries sustained due to
chlorine may be reversed with the addition of appropriate metabolites to the growth
medium (Heinmets et al., 1954).
With reference to the Introduction, the biochemical effect of chlorine upon the
bacterial cell may be found. One specific mode of operation, which was not
mentioned previously, comes from the early work of Knox, et al. (1948) in which it
was proposed that chlorine specifically oxidizes sulfhydryl groups of certain
enzymes irrportant in carbohydrate metabolism. These hypothetical models of chlorine
- 159 -
action prc3vide a basis frcm which the physiologic understanding of this chemical's
function may be extrapolated. This knowledge is essential if chlorine is to be
considered for purposes of sterilization, since its limitations must be known.
Experimentation with chlorine requires the use of acid-washed glassware,
chlorine demand- free test waters and bacteria free of chlorine-demand products.
Fitzgerald and Dervartanian (1969) presented experimental data, regarding P.
aeruginosa, v*iich illustrated vAiy such materials are of paramount iirportance. An
unwashed suspension of P. aeruginosa (concentration of 10E6) had a chlorine demand
of 0.4 ppm v^le washed bacteria had a demand of one- tenth this amount. Some
examples of chlorine-demanding products may again be found within the Introduction.
The reason for their renoval prior to experimentation resides within their ability
to stabilize chlorine and render it nearly functionless .
One final point concerning the operation of chlorine deals with the kinetics
and the more general functioning of this bactericidal agent. Three basic factors
influence chlorine's effect on the bacterial cell. The first of these is the mass
transfer of chlorine to the bacterial cell liquid interface. Secondly, the
chemisorption of the chlorine at selective centres on the cell surface and finally,
"the surface and intrasurface diffusion of the activated chemisorbed complex with
attendant chemical attack on cellular elements" (Bemarde et al., 1967).
This schemata illustrates the "gross" functioning of chlorine, as well, a feel
for the numerous locations for rate limiting steps may be obtained.
The experimental results obtained through this research project (see Results)
seemed to correlate, with only two trial exc^jtions, to the theoretical concepts
presented by Black et al. (1970), in vtiich it was reported that the bactericidal
effectiveness of chlorine was greater at an acidic j^. The inplications here are
- 160 -
that the hypochlorite ion is not as effective a disinfectant as hydrochlorous acid,
v*iich referring to Table 7, makes the adjustment of the pH a prerequisite for
adequate killing.
Table 6
Effect of TpH on forms of Chlorine
pH
%
CI
% HOCl
% OCl
4
0.
.5
99,
.5
5
0
99.5
0.5
6
0
96.5
3.5
7
0
72.5
27.5
8
0
21.5
78.5
9
0
1
99
(Black et al., 1970)
Further examination of the results shows that at higher concentrations the
percentage of organisms killed was increased. The use of chlorine as a sterilant,
however, is questionable. Neither the acidic conditions nor the increased
concentration of 0.4 p^mi (theoretically ideal situation) provided 100% killing,
demanded of an agent used for sterilization. B. breve was the only organism with a
percentage killed close to that of sterility, and it was yet 2 percentage points
short. Further increase in chlorine concentration, coupled with an eiqxssure time
of greater than on minute may be needed to attain sterile conditions.
The final aspect of the chlorine experimentation deals with the ability of some
organisms to resist the lethal effects of this chemical. Four main factors are
- 161 -
associated with this resistance and they include: (i) cell surface structure
mcdification vAiich may facilitate cell clurtping, (ii) bacterial adhesion to
suspended particulate matter (clay particles), (iii) production of extracellular
capsules or slime layers and (iv) formation of resistant spores (Ridgway and Olson,
1982) . Frcm the test organisms used, P. aeruginosa was the only one with the
ability to produce a slime layer which according to Brown (1975) may act by
attaching chemicals and preventing their penetration into the cell. The
experimental results in a pH of 9,0 do, for P. aeruginosa . indicate a lower
percentage killed than the other organisms, hcwever, this was not the case at a pH
of 6.0. P. aeruginosa ' s greater resistance may also be attributed to this
organisms tendency to form cell aggregations.
- 162 -
REOCMMENDATICa^S
6.1 Growth cycle determination
To facilitate the study of the fecal streptococci a superior approach may
be to ccmbine E. coli, P. aeruginosa. B. longum and the first case S.
faecal is var. faecal is and in the second case S. faecium.
The second alteration would be to study the growth of the organisms in an
open-system, as opposed to the closed environment used in this study.
6.2 Chlorine testing
Ihe bactericidal effect of chlorine could be tested on more resistant
organisms, such as the Mycoplasma or endospore forming organisms.
Further experimentation with the manipultion of the time of e>:posure to
chlorine, in conjunction with a wider range of chlorine concentration.
- 163 -
Appendix
7.1 Buffers and Solutions:
1) - Calcitnn hypochlorite (stock) solution:
Ca(OC) 0.5g
dH20 SOOmL
Stir ingredients to dissolve. Refridgerate in the dark.
2) - l.OM Hydrochloric acid:
BDH Analar grade concentrated HCl 41.5iiiL
dH20 SOOmL
I'
Add concentrated HCl to "200mL dH20 and then add the remaining 300inL dH20.
Dispense into bottles and autoclave for 15 minutes at 121°C / 15 PSI. Ke^
ref ridgerated .
3) - Phosphate solution:
(A) Dissolve 34. Og KH2PO4 in 500mL dH20.
Adjust pH to 7.2
Dilute to 1 Litre with dH20
(B) Dissolve 50g MgS04 • 7H2O in 1 Litre dH20.
Autoclave both solutions separately for 15 minutes at 121°C / 15 PSI. Cool and
store for up to 1 month. Add 1.25mL of (A) and 5mL of (B) to 1 Litre of dH20.
Dispense as dilution blanks or for rinse water. Autoclave for 15 minutes at
121°C / 15 PSI.
4) - Sodium thiosulfate solution:
Na2S203 0.35g
dH20 SOOmL
- 164 -
Stir ingredients to dissolve. Add 5inL of Na2S203 to acid washed test tubes.
Autoclave for 15 minutes at 121°C / 15 PSI.
5) - l.OM Sodium hydroxide:
UaCH 40. Og
dH20 lOOmL
Dissolve NaOH in dH20 slcwly. Dispense into bottles and autoclave for 15
minutes at 121°C / 15 PSI.
7.2 Growth Media;
The following media was prepared according to manufacturer's specifications:
DIPXD media:
1) - BHI agar
2) - Nutrient agar 1.5% (NA) 3) -Nutrient broth
4) - Sugars for biochemical testing, prepared to a final concentration of 0.5%.
- Arabinose - Cellcbiose - Xylose
- Lactose - Raf f inose
- Mannitol - Ribose
- Melelitose - Sorbose
- Melibiose - Trehalose
5) - The remainder of the biochemical testing agents were also prepared to
manufacturer's specifications:
- Argenine - Litmus milk - Sodium chloride
- Bile esculin - KLigler's - Todd Hewitt
- Gelatin - Pyruvate
GIBCD media
- 165 -
6) - MRS broth
7) - MRS agar - with the addition of 0.03% cysteine
hydrochloride
8) - M-Enterococcus agar (m-Ent) :
M-Enterococcus agar (DIFCD) 42g
*Sterile dH2 IGOOmL
*Autoclave distilled water first, prior to making media. Allow to cool.
Weigh out agar in a sterile beaker using an alcohol flamed spatula. Heat to
dissolve agar (93°C) . Cool rapidly to 60°C and dispense into square petri
plates. Final pH 7.2 +/" 0-2.
9) - m-E medium (Dufour's modified) :
- Peptone 10. Og
- Yeast extract 30. Og
- Sodium chloride 15. Og
- Sodium azide 0.15g
- Actidione 0.05g
- Agar 15. Og
- dH20 lOOOmL
Mix above ingredients and heat to 90°C. Autoclave for 15 minutes at 121°C / 15 PSI.
Cool medium to 60°C and stir in *Antibiotic solution. Dispense into square sterile
petri dishes. Store at 4°C after solidification. Final pH 7.1 +/~ O-l-
♦Antibiotic solution:
Add each separately, ascepticcilly
A) Nalidixic acid - 0.240g in 3mL dH20
and 0.2mL lOM NaOH
Add last:
- 166 -
B) Triphenyltetrazolium chloride - 0.020g
C) Indoxyl - B - D glucose - O.SOOg in 5inL 95%
ethanol and SmL dH20
10) - Ifedium for Pseudomonas aeruginosa (MPA) :
- L - lysine roonchydroechoride 5,0g
- Yeast extxact 2.0g
- Xylose 2.5g
- Sodium thiosulphate 5.0g
- Magnesium sulphate, anhydrousl . 5g
- Sucrose 1.25g
- lactose 1.25g
- Sodium chloride 5.0g
- Ferric ammonium citrate 0.80g
- Sodium desoxycholate O.lOg
- Phenol red 0.08g
- dH20 sterile 800mL
Autoclave 800 mL distilled water before pr^aaring media.
Mix above ingredients and adjust pH to 7.6. Add 15g agar. Heat to 93°C to
dissolve agar and then cool to 60°C. Stir in *Antibiotic solution. Dispense
into square sterile petri dishes. Store at 4°C after solidification. Final pH
7.1 +/- 0.1.
*Antibiotic solution:
A) Sulfapyridine 0.1760g
B) Kanamycin sulphate 0.0085g
C) Naladixic acid 0.0370g
D) Actidione O.lSOOg
- 167 -
Dissolve A to D in 200inL sterile dH20. Heat to 50°C to dissolve antibiotics.
11) - Medium for the Isolation of Thermo Tolerant E. coli
(M-Tec agar) :
- Proteose p^jtone No. 3
5.0g
- Yeas=t extracrt
3.0g
- Lactose
10. Og
- NaCl
7.5g
- K2HPO4 ; -
3.3g
- KH2PO4
l.Og
- Sodium lauryl sulpiiate
0.2g
- Sodium deoxycholate
O.lg
- Brtsnocresol purple
O.OSg
- Bromo phenol red
0.08g
- Agar
15. Og
- dH20
Mix above ingredients and heat to 90°C to dissolve agar. Autoclave for 15
minutes at 121°C / 15 PSI. Cool to 50°C and dispense into sterile square petri
dishes. Store at 4°C after solidification. Final jSl 7.1 +/- 0.1.
12) - YN17 Blue (Used for the isolation of Bifidobacterium) :
- Yeast extract 20. Og
- Polypeptone BBL 10. Og
- Lactose 10. Og
- Casamino acid 8.0g
- Sodium chloride 3.2g
- Bromocresol green0.30g
- Cysteine hydrochloride 0.40g
- 168 -
- Agar 15. Og
- dH20 lOOOltlL
Mix above ingredients on medium heat until agar dissolves. Autoclave for 15
minutes at 121°C / 15 PSI and then cool to 60°C. Stir in *Antibiotic solution.
Dispense into sterile petri dishes. After solidification store at 4°C. Final
pH 6.9 +/- O-l-
*Antibiotic solution:
A) Nalidixic acid 0.03g
B) Kanaycin sulphate 0.05g
C) Polymixin B 0.0062g
13) - Growth media for Bifido YN-17
- Follow same procedure as above, however, emit bromocresol green and
antibiotic solution
- 169 -
REFERENCES
American Public Health Asscxriation, American Water Works
Association, and Water Pollution Control Federation. 1971. Standard Methods
for the Examination of Water and Wastewater. American Public Health
Association, Inc. , 13th ed. , New York.
Bemarde, M.A. , Snow, W.B. , Olivieri, V.P. and Davidson, B. 1967.
Kinetic and Mechanisms of Bacterial Disinfection by Chlorine Dioxide. Applied
Micrctoiology. 15: 257-265.
Black, A.P. , Kintnan, R>N. , Keim, M.A. , Smith, J.J. and Harlan,
W.E. 1970. The Disinfection of Swimming Pool Water. Pari I. Conparison of
Iodine and Chlorine as Swimming Pool Disinfectants. American Journal of Public
Health. 60: 535-544.
Black, A.P. , Keim, M.A. , Smith, J.J., Sykes, G.M. and Harlan,
W.E. 1970. Ihe Disinfection of Swimming Pool Water. Part II. A Field Study
of the Disinfection of Public Swimming Pools. American Journal of Public
Health. 60: 740-750.
Brodsky, M.H. and Schiemann, D.A. 1976. Evaluation of Pfizer
Enterococcus and KH Media for Recovery of Fecal Streptococci from Water by
Membrane Filtration. Applied and Environmental Microbiology. 31: 695-699.
*Brown, M.R.W. 1975. The Role of the Cell Envelope in
Resistance. Resistance of Pseudomonas aeruginosa , John Wiley and Sons: New
York. (Ch. 3) .
*Canper, A.K. and McFeters, G.A. 1979. Chlorine Injury and the
Enumeration of Waterbome Coliform Bacteria. Applied and Environmental
Microbiology. 37: 633-641.
Doran, J.W. and Linn, D.M. 1979, Bacterial Quality of Runoff
Water from Pastureland. implied and Environmental Microbiology. 37: 985-991.
Fitzgerald, G.P. and DerVartanian, M.E. 1969. Pseudomonas
aeruginosa for the Evaluation of Swimming Pool Chlorination an Algicides.
Applied Microbiology. 17: 415-421.
Geldreich, E.E. and Kenner, B.A. 1969. Concepts of Fecal
Streptococci in Stream Pollution. Journal of Water Pollution Control
Federation. 41: 336-352.
Gibbons, N.E. and Buchanan, R.E. 1985. Bergey's Manual of
Determinative Micaxbiology. The Williams and Wilkins Co.: Baltimore.
- 170 -
Gyles, C.L. 1984. Environmental Aspects of Escherichia coli in
Human Health. National Research Council of Canada. NRCC. No. 22490.
Heinmets, F., Taylor, W.W. and Lehman, J.J. 1954. The Use of
Metabolites in Restoration of the Viability of Heat and Chemically Inactivated
E. coli. Journal of Bacteriology. 67: 5-12.
Isenberg, H.D. , Goldberg, D. and Saitpson, J. 1970. laboratory
Studies with a Selective Enterococcus Medium. Applied Microbiology. 20: 433-
436.
Knox, W.E., Stunpf, P.K. , Green, D.E. and Auerbach, Y.H. 1948.
The Inhibition of Sulfhydiyl Enzymes as the Basis of the Bacterial Action of
Chlorine, Journal of Bacteriology. 55: 451-458.
Lee, Wie-Shing. 1972. Improved Procedure for Identification of
Group D Enterococci with Two New Media. Applied Microbiology. 24: 1-3.
Levin, M.A. and Cabelli, V.J. 1972. Membrane Filter Technique for Enumeration of
Pseudomonas aeruoginosa. ^^plied Microbiology. 24: 864-870.
Levin, M.A. , Fischer, J.R. and Cabelli, V.J. 1975. Membrane
Filtration for Enumeration of Enterococci in Marine Waters. ;^plied
Microbiology. 24: 66-71.
McFeters, G.A. . Bissonnette, G.K. , Jezeski, C.A. , Thomson, C.A.
and Stuart, D.G. 1974. Cortparative Survival of Indicator Bacteria and Enteric
Pathogens in Well Water. Applied Micrcbiology. 27: 823-829.
Ridgway, H.F. and Olson, B.H. 1982. Qilorine Resistant Patterns
of Bacteria from Two Drinking Water Distribution Systems. ;^plied and
Environmental Microbiology. 4: 972-987.
Ringen, L.M. and Drake, C.H. 1952. A Study of the Incidence of
Pseudomonas aeruginosa from Various Natural Sources. Journal of Bacteriology.
64: 841-845.
Schuettpelz, D.H. 1969. Fecal and Total Coliform Tests in Water
Quality Evaluation. MSc. Ihesis. University of Guelph.
Seyfried, P.L. and Eraser, D.J. 1980. Persistence of Pseudomonas
aeruginosa in Chlorinated Swimming Pools. Canadian Journal of Microbiology.
26: 350-355.
Seyfried, P.L. 1988. University of Toronto. PersoncLL
Communication.
Switzer, R.E. and Evans, J.B. 1974. Evaluation of Selective
Media for Enumeration of Group D Str^stococci in Bovine Feces. Applied
Microbiology. 28: 1086-1087.
Taylor, R.H. , Bordner, R.H. and Scarpino, P.V. 1973. Delayed -
- 171 -
Incubation Membrane - Filter Test for Fecal Coliforms. ^plied Microbiology.
25: 363-368.
Wheater, D.W.F., Mara, D.D. , Lozan Jawad and Oragui. 1979. Journal of
Biological Indicators of Waste Quality, John Wiley and Sons: New York (Ch.
21)
- 172 -
APPENDIX C
THE ISOIATICN AND lUtNl'lflCAnCN
OF HEFIDCffiftCEERrA FPCM FBCAL
AND SEHAGE SAMPLES
In-ja Huh, Department of Microbiology
University of Toronto
- 173 -
TABLE OF aCNEENTS
Page No.
ABSTRACT ^'^'^
INTRDDUCnON I'^S
OBJECnVES ISO
MATERIAI5 AND METHODS 181
Collection and preparation of the test sattples 181
Analysis of samples 181
Identification of the isolates 182
Carbohydrate fermentation profile 183
Flash freezing 185
RESULTS 185
DISCUSSION ■ 154
CONCLUSIONS 196
RECCMMENDATIONS l^'^
APPENDIX 158
REFERENCES
202
- 174 -
T.T57r OF TABCZS
Table Page No.
C-1 The carbohydrate fermentation profile used to 184
speciate the bifidobacteria isolated from the
fecal and sewage sanples
C-2 Geometric mean concentration of FC, E^ coli 187
and bifidobacteria in the storm and sanitary
sewage samples
C-3 Geometric mean concentrations of E^ coli ' 189
and bifidobacteria in the feces of humans,
cats and dogs
C-4 The carbohydrate fermentation reactions 190
of ATCC cultures
C-5 The biochemical test scheme used for the 191
identification of bifidobacteria in the
profiles of ATCC cultures and their
Gram Stain characteristics
C-6 Species of bifidobacteria isolated from 192
the two selective media that occurs in
fecal and sewage sanples
- 175 -
LIST OF FIGURES
Figure Page ^Jo.
C-1 Geometric mean concentration of PC, E^ coli and 188
bifidobacteria in the storm and sanitary sewage samples
C-2 A conparison of the recovery of bifidobacteria species 193
from feces and sewage on two selective media
C-3 Percentage species of bifidobacteria in feces, sanitary 195
and storm sewage
- 176 -
LIST OF ABBREVIATICNS
° C = Degree Celsius
FC = Fecal coliforms
G = Grains
hrs = Hours
HBSA = Human bifid sorbitol agar
i.e. = That is
L = Litres
LiCl = Lithium chloride
MF = Membrane filtration
mg = milligrams
mL = Milliliters
min = Minutes
mm = millimeters
m-Tec = Medium for thermotolerant Escherichia coli
nm = Nanometres
s = seconds
YN-17 = Medium developed by Mara and Oragui for the enumeration of
bifidobacteria
- 177 -
ABSTOACr
The distribution of Bifidobacteria in the environment was studied
utilizing two selective media: YN-17 and HBSA. The feces of humans, cats and
dogs as well as sanitary and storm sewage samples were analyzed and
Bifidobacteria were isolated in all cases except for the dogs. Species of
sorbitol-fermenting Bifidobacteria, which have been previously reported as
being exclusive to human hosts, were isolated from feces of human and cats as
well as from sanitary and storm sewage sanples. However, 100% of the isolates
from cats did not ferment sorbitol v*ien a carbohydrate profile was carried out.
These sortitol-fermenting species were recovered using both YN-17 and HBSA.
However, a higher percentage recovery was made on HBSA as this medium is
specific for sorbitol-fermenting species. The results of this study indicate
that sorbitol-fermenting Bifidobacteria may be useful as indicators of recent
human fecal pollution in surface waters.
- 178 -
DracDDcrrcN
cue to the disease hazards associated with fecally-contaminated water, it
is imperative that the presence of fecal wastes be rapidly and accurately
detected in surface water bodies, and the sources of these inputs traced in
order to eliminate them. Thus a reliable bacterial indicator is desired which
will detect recent fecal pollution and will in some way differentiate the
source of the input (i.e. human and non-human) . . . .-.
In 1981, the Toronto Area Watershed Management Study was initiated to
better define the water quality conditions in Humber, Black Creek, Don Rivers
and along Toronto Beaches. Particular emphasis was placed on urban areas of
Metropolitan Toronto especially on point pollution inputs since high levels of
fecal indicating bacteria were found in storm sewages. Subsequent studies
began in the fall of 1982 to assess the contribution of pollution loading by
the storm sewers and to trace the source of pollution in these lines.
Unfortunately, pinpointing the original source of pollution is difficult
because a specific bacterial indicator of human fecal wastes is not available
at the present time.
Traditionally E^ coli and FC have been used as the indicators of fecal
pollution in storm waters and surface waters but due to its wide distribution
in both human and animal feces they are not acceptable as an indicator of human
input only. A number of workers have suggested using Bifidobacterium as a
fecal indicator. (Buchanan and Gibbon, 1947; Scardovi et al, 1971; Levin, 1977
and Resnick and Levin, 1981) .
The genus Bifidobacterium includes gram-positive, non-sporulating, non-
motile, anaerobic, pleomorphic rods. At the present time, there are 24 known
species of bifidobacteria that have been identified. Bifidobacteria are
- 179 -
present in concentration of 10^ organisms per gram of feces in humans
(Geldriech, 1978) , but have a very limited distribution among other animals
(Mara and Oragui, 1983) . They also have been recovered from raw se'.^rage
(Resnick and Levin, 1981) and in a Tropical Pain Forest Watershed B.
adolescentis was isolated and enumerated. (Carillo et al, 1985) .
The first synthetic selective medium that gave a reasonable recovery of
bifidobacteria was invented by Gyllenberg and Niemen in 1960 and was called GN-
6. This was modified in 1981 by Resnick and l£vin Vi^o added nalidixic acid to
(3^-6 and named it YN-6. Ihen, in 1983, Mara and Oragui developed YN-17 medium
which is more selective for bifidobacteria due to a further modification of YN-
6 (i.e. addition of polymyxin B and kanaraycin sulphate) . These antibiotics
decreased the streptococcal contamination in the selective medium but did not
eliminate them.
In that same study, Mara and Oragui proposed a methodology to detect
bifidobacteria species as indicators of human fecal wastes in surface waters.
They devised a medium called HBSA which is very similar to YN-17 except its
replacement of fermentive carbohydrate lactose by sorbitol. Their method
involved the isolation of bifidobacteria by membrane filtration of test sanples
onto HBSA v^ch only allowed growth of sorbitol-fermenting species. This study
reported that sorbitol-fermenting species of bifidobacteria were exclusive to
human fecal wastes. These species were B^ adolescentis and B^ breve .
- 180 -
c«jBcrrvEs
The purpose of this project was:
To test the relative merits of HBSA in conjunction with another standard
medium for the isolation and enimieration of bifidobacteria which is YlJ-17.
To study the possible usage of MRS medium as a selective medium for
bifidobacteria and test the effectivity of LiCl in repression of
streptococcal contamination.
Isolation and speciation of bifidobacteria found in feces of humans, cats
and dogs as well as storm and sanitary sewage samples.
To confirm vrtiether or not sorbitol-fermenting species are found in human
waste but are absent in animal wastes.
To compare the concentration of fecal coliforms and E^ coli with
bifidobacteria count in feces and in sewage samples.
- 181 -
MATERIALS AND MEIHDDS
Oollecticn and Pr^araticn of the Test Sanples
In the fall of 1987 samples were collected from three points along a storm
sewage line draining the Danforth-Pape Area and cilso from two points along a
sanitary sewage line located in proximity to the storm water. The samples were
collected in triplicates for statistical accuracy. This particular storm sewer
line was chosen because it was suspected of having an illegal sanitary hook-up.
Water samples were transported to the laboratory on ice in sterile sampling
bottles with sodium thiosulphate to neutralize any chlorine that might be
present.
Fecal sanples were collected in presterilized wide-mouthed jars. A total
of 20g of feces was weighed out, diluted 1 in 10 with sterile buffered
phosphate water and mixed in a blender for 15s at low speed before being
examined. All the samples were analyzed within 2-4 hours of its collection.
Analysis of Sanples
Both the sewage and fecal samples were tested by membrane filtration
analysis. This method is one of the standard processes used in water quality
monitoring (Cufour and Cabelli, 1975; Dufour et al, 1981). Serially-diluted
samples were filtered through Gelman 0^-6 47mm nitrate filters having a
porosity of 0.45um. A control sample of sterile buffered water was filtered
for each of the samples by placing these filters on the least selective medium
(i.e. m-Tec) . The filters of the sanples were planted on the selective media
for the recovery of fecal coliforms and bifidobacteria.
FC bacterial densities were determined by using m-Tec agar which was
incubated at 44.5 +/~ 0-5° C for 23 +/" 1 h^^- The target colonies enumerated
- 182 -
were yellow, yellow-green and yellow-brown on this medium. To ensure the
accuracy of the counts, filters with 10 - 100 target colonies were chosen to
enumerate. These filters were subsequently tested for E^ coli confirmation
using the urease test. The test involved placing the filter on a pad soaked in
a urea phenol red solution and allowed to react for 15 minutes. Deamination by
non-E. coli coliform bacteria possessing urease causes a colour change in the
colonies from yellow to pink. A second count of urease negative (yellow)
colonies was then taken.
The isolation and enumeration of bifidobacteria was carried out using YN-
17 and HBSA selective media. These media were incubated anaerobically in jars
with hydrogen and carbon dioxide gas packs for 48 hrs at 37° C. The target
colonies on the YN-17 medium were characteristically circular, convex, mucoid,
2-3inm in diameter, glistening, greenish-blue and dark blue and on HBSA they
were brownish-yellow to yellow. Streptococcal contamination on W-17 was
exhibited by the presence of the greenish-blue colonies with pale green
periphery an on HBSA by light yellow and clear colonies. These colonies were
not enumerated and were considered as background. For the formulation of the
media used see Appendix.
Identif icatiai of Isolates
From the total count of bifidobacteria that exhibited the typical target
morphology on the selective media, 10% were isolated to be identified. These
isolates were streaked out on a growth medium for purity. The growth medium
used was YN-17 base without the antibiotics and the indicator. After
incubation these isolates were Gram stained and those rods with typical
pleomorphic morphology (i.e. bifurcated v an Y forms, club shaped ...) were
- 183 -
Streaked out for the second time on the growth medium for purity and incubated
both anaerobically and aerobically. Isolates exhibiting either gram-negative
rods or gram-positive cocci morphology were discarded. Only isolates which
grew anaerobically were Gram stained once again and those with typical pleomorphism
were considered as bifidobacteria.
The confirmation of bifidobacteria was further verified by carrying out
the following biochemical tests: catalase, gelatin and arginine, Kliger's ircn
and bile esculin. Then the speciation of the bifidobacteria isolates -.vas
accoiiplished by observing the fermentative reactions of the isolates in nine
different sugars. The typical fermentation patterns of Bifidobacterium species
is illustrated in Table 1.
Carbohydrate Fermentation Profile
The carbohydrate media was made by dissolving 8.0g of phenol red broth
base in 450 mL distilled water. A filter sterilized sugar solution was added
to this autoclaved broth to give final concentration of 1%. The sugars used
were the following: L(+) Arabinose, D-Cellobiose, D-Mannitol, D(+) -Melezitose,
D-Ribose, D-Sorbitol, soluble starch and D(+)Xylose. The test tubes of car-
bohydrates were incubated for 24 hrs at 37 ' C before the inoculation of the
isolates in order to reduce oxygen tension and to ensure sterility. The inoculated
tubes were read after anaerobic incubation of 48 hrs and if the results were
not definite, they were reincubated for an additional 24 hrs.
- 134 -
[•able C-1
Tat]e !. The carbohydrate fermentation reaction profile used to
sped ate the Bifidobacteria isolated from the fecal and sewage
samples. { formulated from the Bergeys tlanual }
BlfldODaCt.
Arabnose
Cellobiose
Lactose
Mannitol Mele:itose
Ri bote Sorbitol
Starc-h >
'.ijIct-
species
bifiijum
-
-
+
-
-
-
-
lonaum
+
-
+
+
+
-
V
infanti?
-
-
+
-
+
-
V
bri=">'^
-
V
+
V V
+ V
-
-
8doli??ci?nti3
+
+
+
V +
+ y
•♦•
■^
ana upturn
+
-
+
-
+ V
+
+
catrnui-tiTijm
+
+
+
i
V
+ *
-
•r
P?ir'jdOC2tnijlatU
m +
V
+
-
+ V
+
+
dentiijrn
+
+
+
+ +
+
+
f
qlcibosum
V
-
+
-
+
+
V
psijijdolciriqijm
+
V
V
V
+
+
■k
cunii.-un
+
-
-
-
-
+
+
choenrnjm
-
-
+
-
-
•^
-
ariirri.'jil;.
+
V
+
Y
+
+
•T
trirrTioC'Ciilum
-
V
V
V
-
+
-
bourn
-
-
V
-
-
+
-
maoriijrri
+
-
+
-
+
-
■^
pullorum
+
-
-
-
+
-
+
3IJV?.
+
-
+
-
-
-
■4.
minirnij-n
-
-
-
-
-
^
-
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-
-
-
+
+ J-
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-
corurif-'''orme
+
+
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+
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-
aStenoOt";
+
+
-
-
+
-
f
indicijrn
+
+
"+"= The sugar was fermented
■-"= The ?ugar va? not fermented
■v'"= Some strai n? ferment this sugar
- 185 -
Flash Freezing
The ATCC cultures were flash frozen for long-term storage. The cultures
were prepared by growing on a growth medium for purity and quantity. Then
under the Biohazard Hood, approximately 5 mL of 40% glycerol was poured onto
each of the plates and with a flamed loop the colonies were dispersed. By
using 1 mL pipettes the culture-glycerol solutions were soaked into small
pieces of filter papers v*iich had been aseptically placed in labelled freezer
bags. These were done in triplicates and were heat-sealed. The bags were
placed in a small box and stored in a -70° C freezer.
The frozen cultures were revived by planting the saturated filters on the
growth medium and streaking for isolated colonies.
Results
The YN-17 medium allowed more accurate enumeration and isolation of
bifidobacteria than HBSA as indicated by the higher percentage of false
positive targets (i.e. 10% for HBSA and 5% for YN-17) . A large majority of
these false positive targets were fecal streptococci. MRS agar with the
antibiotic cocktail of YN-17 was also tested for its selectivity of
bifidobacteria. Unfortunately, this medium supported growth of both
streptococci and Lactobacillus species which resulted in higher concentrations
of background bacteria. However, this medium shortened the incubation period
by half and thus was used without the antibiotics as a growth medium. In
addition, LiCl was incorporated at the concentrations of 0.3%-0.4% to YN-17 and
HBSA; however it did not decrease the streptococcal contamination as was
suggested by N.A. KLapes at the ASM meeting in March of 1987.
- 186 -
The geometric mean concentrations of FC, E^ coli and bifidobacteria in storm
and sanitary sewage samples can be seen in Table 2. Also, a representative
graph of the counts from storm sewage samples is illustrated in Figure 1.
Both Table 2 and Figure 1 indicate that in storm sewers total bifidobacteria
exist in hic^er concentrations than FC or E^ coli. The table and figure also
show the trend towards decreasing concentrations of all the bacteria from
points A to C due to dilution and die-off within the storm sewer line. From
the graph one can clearly see that in storm se'^rage bifidobacteria exist in
concentrations about 10 times greater than E^ coli . Note that there are
higher concentrations of bacteria in sanitary se'.:rage than in stonn sewage and
that variance in bacterial counts occurs over the different sample days.
Results of the fecal analysis (Table 3) also illustrates a higher concentration
of bifidobacteria than E^ coli (i.e. 10 - 100 times greater). Bifidobacteria
were isolated from the feces of humans and cats but not from dogs. The sewage
samples had lower concentrations of bifidobacteria and E^ coli than the fecal
samples. This is due to dilution and to the fact that the organisms die off at
a faster rate in the environment (Oragui, 1982) .
The fermentative reaction of ATCC cultures of bifidobacteria (Table 4)
closely corresponds with those of the Bergey's Manual profile used for speciation
of the isolates (Table 1) . Table 5 illustrates the biochemical reactions used
to confirm bifidobacteria (Bergey's Manual 9th ed.) . The ATCC cultures exhibited
typical biochemical reactions for this genus (i.e. catalase (-) , arginine (-) ,
acid production in Kliger's iron medium and variable hydrolysis of bile esculin) .
Recovery of bifidobacteria species from feces and sewage on two selective
media is shown in Table 6 and Figure 2. A wider variety of bifidobacteria
- 187 -
Table C-2
T^i^/e 2. osametnc mffs/? cance/itrdifa.'js of fees' Cij/!forj7?s.
Esc^anc^/s ra// and Bffidcbscierld In the storm and ssnftsru
( count;: oef 1 Ou mi of i^^rriDie )
( 1 9S6) its -SOU re* FC EC HESm 'iU-\
Oct Z: A 3933 3617 ■ - 1^-66
Oct 26 A 10Z33 10200 133000 27000';
Nijv 13 A 39666 57500 21000 46000'
3 26000 23700 1600 52000
C 1 300 030 1 500 -000
Oct 2o D 253000 ■ 220000 - - 96000
Mcv 13 D 236 6000
220000
-
1 700000
f 7 ''! !'! r
2600000
4700::
E j7:.0iJiJU
•J;2ri31 E - 2900000 42000
'v'N-17 = Tijt.ii count of EifidOuoCtena
H5'£a = Luunt of t.hi; iorDitoI ferment; no Bifidotj.icfen.^
- 133
»— i
—I
O
CJ
LU
cn
O
Ll_
O
<:
CJ
LU
Li.
Ll_
O
CD
LU
CJ
O
CJ
LU
CJ
I— I
tx
LU
O
LU
CD
LU
CD
LU
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a:
a
CL
LU
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a
Ll_
I— t
02
a
^ >•
i
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LU
mo: 4.
o as —
a =
cn I
_1 LU
O 1
u
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II LU
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u u.
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U
LU ,
t 1
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<-
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03
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22
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(^W00;/W9) M0IiY«LNC3 "VIHrlGYS
139 -
Table C-3
TaifiB J. Seomstric mas/? csncsnirdiians of ^•r.c.^.^^;r.?;.^ ,-.?.-/ art
Sifidoiydcisria in tf^s faces af humans, cats and dags.
S cure 8 EC H5Sa VN- \ 7
( per I gr:m -jf r'^csi )
human 230.000,000 20,000,000 37.000 O'j-O 0
cat 6,400,000 17,000,000 526.000,000
dog 3,067,000 0 0
- 'J30 -
Table C-4
Tj^/a 4. The chardci eristic ferinenisti'/e reset ions of 9 eugere
by the A TCC cultures of Sifidottacteria.
LiirjM.Ti
iriT.KiTI!
g!^r''';lor'jm +
hrr^''*
ir^nlrifprl'ltl ;: + +
:ini3ij,vi
- 191 -
Table C-5
TatJe 5. The bfochemica} issi scheme used for the
fdentificatjon of Bifidohacieria in the profiles of A TCC culiur
and their Sram Stain Characteristics.
rrior'Dhc-loQ^j hudrolgsiJ hydro. h yd ''■::. ir:n
I ^ ■»■ ~ f J L' ' ' I 'J '.i u I
pliromorphic
"■irripnor.-j-
■ ike "Cell::;
1^697 inf:5riti:5
G +v*
pltornorphic
1 56 9S Dyrvulonjm
G +ve
branch! nq,
lorig, ti-iui
rods
15700 tifrvf'
G +ve
15701
thin.:?: .5 tort
rods
15702!1!je!-orurri
G +ve
loni] .i]^. short
thin rod?
15703.iijuir::.i::'!'nt:v
G -^ve
1 57 0~ piwrnorphio
having many of
other spirci's'
morphology
1 5707 long urn G +ve
long cc'lls v/ith
oiub ihapird ends
'"^" - ir-'id produc'ion
■'-■■= Hydrolysis v:3s odSc'rvi?d
■■-"= Hudrolijjv? v.':s not obssrv^Jj
- 192 -
Table C-6
Tall I a D. Species of difiuOudctsha isoJdted from the tYr-a
ssJectfve media that occurs in fecal and sewape samples.
3pc;C;r.;5
j^:c^'r■^c^^^t1i; wuman 6/9 3/ 1 6
«t
jtorm sev^jQr - 4/4
jynit^fu JcVtag* 1 ! /' i 1 O/I'i
'one 'J"; riijn-ian l.-'9 7/16
storm
brr'-r fiurnsn 2/9 l/lo
cat , 12/13
storrn
sanitary - 3/19
gli^bcsum hurrian
cat
storm
sanitary 1/13 ' 1/19
D-re-iJcoc-atnji.-jtijm human
car 1 / 1 3
storm i/i
sanitary
suis human
cat
storm
sanitary - 1/19
ariQijii"'."' human
oar 1/13
storm
sanitary 3/li 2/19
thr-fTr;i;c;h;iijm nuTian
cat 1 / 1 3
sTorm
sanitary
'^I'lorium humian
cat
storm
sanitary
uniiientTTViie human
cat 3/ i 3
*tocm
iamtjr-'j 2/13 2/19
- 193 -
O
d
cn
I— I
u
LU
Q- <
cn H-(
a
<: LU
cr I
QJ LLi I
h- >
< I—
CD CJ
O IXl
a —I
j—l LU
Ll_ 01
m o
U- I—
o
:^ o
CL
LU UJ
> CD
O <
O S
UJ LU
cc o:
LU a
< ■
LU I
a
cn
I— •
cc
-<
Q.
O
CJ
UJ
<
in
cn-
PS
LU
C3
■<
LU — rr
cnr*
>- 1
crz
<>-
•<
cn
cn
LU
CJ
Lu:
cn
CJ —
Lur-
zz
■< >-
I
^^s
cr
<4..
I I I I I
5fi:TC:>iV»:*f:*-
%
r
^?????ff??t^^¥s^
cn
LU
hH
U
LU
a.
cn
<
1/0
o
! ^
<
\
1
'd.
<
y.
(N
<$>
J,
P
H
•H
■>*.
%
53N=Hn330 39ViN53U3d
c?
- 194 -
species were recovered in the sewage samples than in fecal sairples (Fig. 3) .
Figure 3 illustrates that the predcminant species in human feces and se'^rage
were B^ adolescentis and B^ breve which are the sorbitol-fermenting species of
bifidobacteria (Mara and Oragui, 1983) . As indicated by Fig. 3 B^ breve was
isolated from cat samples but 100% of these isolates did not ferment sorbitol
when the carbohydrate profile was carried out.
Discussicn
The superiority of the two selective media is due to the antibiotic,
kanamycin sulphate, which reduced levels of the contaminating streptococci
greatly v^en used in the specified concentration. However, it didn't
completely eliminate the background of streptococci. Yield of bifidobacteria
was higher on YN-17 than PiBSA because HBSA was a more selective medium in that
it only allowed growth of sorbitol-fermenting species. In addition, target
colonies were more easily distinguished on YN-17, while there was a tendency
towards more false (+) targets arising from streptococci on HBSA.
The data presented herein disagreed with the study done by Mara and Oragui
in 1983. In this project it was discovered that their description of
Bi f idobacterium on YN-17 (i.e. blue centre with pale green periphery) was in
fact a mixture of bifidobacteria and streptococci. Thus, during the beginning
of the year the successful recovery rate of bifidobacteria was considerably
lower; however, accuracy of 95% and higher was achieved during the latter part
of the year.
Bifidobacteria can be used as fecal indicating bacteria because they exist
in high number in both feces and fecally contaminated environments. They also
135 -
f I I
a
•z.
<:
>-
<:
en
en
LU
CJ
LU
Ll.
LU 5
en
CD
U-
o
en
LU
I— I
CJ
LU
a.
en
LU
eD
ILU
cr
LU
C
o
C31
UJ
■<
X
Ui
en
c
"^
CO
Ui
cs
<
UJ
en
>•
cr
■<
•<
03
\
\
^
>i.
^
H'
SJ
d
Essss:
[Id
en
UJ
u
UJ
u.
z
I
%
IP.
>.
\\\\\\\\\\\\\\\\^\\^y\\\\^\'^■
o
IS
en
u
UJ
a.
en
! C3
z
<
I <:
■ s
i 3
1 -^
I '-J
I <
a.
m
I
b
S3N2fcinC30 39ViN=2UHd
- 196 -
do not multiply outside the body and a rapid methodology for recovery from the
aquatic environment is available as illustrated in this study.
Site A of the storm sewer line is the likely location of the illegal
connection since the concentration of fecal indicating bacteria was highest at
this in-line point. The illegal hook-up is also confirmed by the presence of
sorbitol-fermenting bifidobacteria in the line. Sanitary sewage samples
contained a wide variety of species of Bi f idobacterium which is possibly due to
some inputs of animal wastes as well as humans, but the highest recoveries made
were again of sorbitol-fermenting bifidobacteria and this correlates with the
face that human feces mate up the greatest percentage of wastes in sanitary
sewage. Although bifidobacteria can be recovered from cats, it is unlikely
that cat feces represent a major source of fecal contamination in the storm
sewer line. Therefore sorbitol-fermenting species can be used to monitor
recent human fecal contamination in sewage lines.
Oonclusicns
* YN-17 yielded higher concentrations and represented a broader spectrum of
different species of bifidobacteria than HBSA.
* B^ adolescentis was the most prevalent species of bifidobacteria in human
feces and sewage samples, but were absent in cats.
* Sorbitol-fermenting species (i.e. B^ breve) were isolated from both human
and cat feces but 100% of the isolates from the cats didn't ferment
sorbitol when the carbohydrate fermentation profile was carried out.
* Bifidobacteria counts were 100 times greater than E^ coli in feces and 10
times greater in sewage sanples.
- 197 -
Recannendaticns
* Further study to confirm the specificity of the selective media for
bifidobacteria.
* Carry out a large scale fecal sairple study to see if the sorbitol-
fermenting species, B^ adolescentis , is predominant in human feces and is
limited or absent in animal feces.
Acknowledgements
The author would like to sincerely thank Elizabeth Harris and
Dr. P.L. Seyfried for their support throughout the year.
- 198 -
1. Dissolve 34. Og of KH2PO4 in 500 mL distilled water and adjust the pH to
7.2. Then dilute this to IL by adding distilled water.
2. Dissolve 50 g of M3SO4 7H2O in IL of distilled water.
These two solutions were autoclaved separately for 15 min at 121° C. After
these were cooled to room temperature, add 1.25 mL of 1) and 5 mL of 2) to XL
of distilled water. Then it can be dispensed either as dilution blanks or as
rinse water for M.F. and autoclave again at 121° C for 20 minutes.
- 199 -
m-Tec TWgar
This medium is used to isolate and enumerate the Fecal Col i forms as well
as the Thermo-tolerant Ej. coli.
Proteose peptone #3 10.00 g ■
Yeast extract • 3.00 g
Lactose 10.00 g
NaCl 7.50 g
K2HPO4 3.30 g
KH2PO4 1.00 g
Sodium lauryl sulphate 0.20 g
Sodium deoxycholate 0.10 g
Bromocresol purple 0.08 g
Bromophenol red 0.08 g
Agar 15.00 g
Distilled water 1.00 L
The above ingredients were mixed and heated to dissolve. Autoclaved for
15 min at 121° C and dispensed into Petri dishes after it is cooled. Before
pouring the pH is adjusted to 7,1 +/~ O-l- '
- 200 -
Bie two selective media for Bifidobacteria
Yeast extract
Poly-peptone (BBL)
Lactose
Casamino acid
NaCl
Bromocresol green
Bromocresol purple
Cystein hydrochloride
Agar
Distilled water
The above ingredients were mixed in a 6L beaker and were stirred on medium
heat until agar was dissolved. Then it was autoclaved for 15 minutes at
121" C. After the isolation was cooled to 60" C the following were added:
Nalidixic acid 30.0 mg 20.0 mg
Kanamycin sulphate 50.0 mg 50.0 mg
Polymixin B 6.20 mg 1.20 mg
Finally, the pH was adjusted to 6.9 +/- 0-1 a^ then the media were poured
into square Petri dishes (100 on^) .
YN-17
HRSA
20.00
g
20.00 g
10.00
g
10.00 g
10.00
g
10.00 g
8.00
g
8.00 g
3.20
g
3.20 g
0.30
g
-
-
0.10 g
0.40
g
0.40 g
15.00
g
15.00 g
1.00
L
1.00 L
- 201 -
Distinct Gram MorphDlogies that are Species Characteristics of Bifidobacteria
Bifidobacterium species
Gram morphology
bifidum
loncrum
breve
ancrulatum
catenulatum
qlobosum
animal is
hi<^ily variable but has the typical
"airphora-like" shaped cells
very elongated and relatively thin
cell with slightly irregular contours
and rare branching
thinnest and shortest rods
v( angular) or palisade arrangement
similar to those of corynebacteria
chains of 3, 4 or more globular
elements
distinct branching
short, coccoid or almost spherical to
curved or tapered
arranged in singly or doubly or rarely
in short chains
characteristic central portion slightly
enlarged
- 202 -
ReferEncjes
1. Ali, M. , Murray, P.R. , Sondag, J.E. 1979. Relative Recovery of anaerobes
on different isolation media. J. of Clinical Microbiol. 10(5) .
2. Cabelli, V.J. , Dufoixr, A. P., McCabe, L. J. , Levin, M.A. 1983. A marine
recreational water quality criterion consistent with indicator concepts
and risk analysis. J. of Water Poll. Cont. Fed. 55: 1306-1314.
3. Carillo, M. , Estrada, E. , Hazen, T.C. 1984. Evaluation of
Bifidobacteria as a possible indicator of human fecal contamination in
tropical fresh-water. Abstract from the Annual Meeting of the American
Society of Microbiology, March 4-9, 1984.
4. Carillo, M. , Estrada, E. , Hazen, T.C. 1985. Survival and enumeration of
the fecal indicators Bi f idobacterium adolescentis and Escherichia coli in
a tropical rain forest watershed. ^^1. Environ. Microbiol. 5 0(2): 468-
476.
5. Cufour, A. P., Strickland, E.R. , Cabelli, V.J. 1981. Membrane filter
method for enumerating E^ coli. Appl. Environ. Microbiol. 41(5): 1152-
1158.
6. Cufour, A. P., Cabelli, V.J. 1975. A membrane filter procedure for
enumerating and component genera of the coliform group in seawater. Appl.
Microbiol. 29: 319-833.
7. Essers, L. 1982. Simple identification of anaerobic bacteria to genus
level using typical antibiotic susceptibility patterns. J. of Appl. Bact.
52: 319-323.
8. Geldreich, E.E. 1970. Applying bacteriological parameters to
recreational water quality. J. Amer. Wat. Works Assoc. 62(2): 113-120.
9. Geldreich, E.E. 1978. Bacterial populations and indicator concepts in
feces, sewage, stormwater and solid wastes.
10. Gyllenberg, H. , Niemella, S., Sormunen, T. 1960. Survival of bifid
bacteria in water as compared with that of coliform bacteria and
enterococci. Appl. Microbiol. 8:20.
11. Mara, D.D. , Oragui, J.I. 1983. Sorbitol-fermenting bifidobacteria as
specific indicators of human fecal pollution. J. of Appl. Bact. 55: 349-
357.
12. Mutai, M. , Tanaka, R. 1980. Improved medium for selective isolation and
enumeration of Bifidobacteria. Appl. Environ. Microbiol. 40(2): 866-869.
13. Oragui, J.I. 1982. Bacteriological methods for the distinction between
human and animal fecal pollution. Eh.D. Thesis, University of Leeds,
England.
14. Resnick, I.G., Levin, M.A. 1981. Quantitative procedure for enumeration
of Bifidobacteria. Appl. Environ. Microbiol. 42(3): 433-438.
- 203 -
APEBOUX D
A STUDY OF "fflE SURVIVAL OF BIFIDOBACIERIA AND
"fflEIR RDLE IN WKEEK QUALITY CUNIMDL
Sheila Shibata, Department of Microbiology
University of Toronto
- 204 -
TABLE OF OCNTENIS
Page No.
LIST OF TABLES 205
LIST OF FIGURES 206
LIST OF ABBREVIATIONS 207
INTRDDUCnON 208
OBJECTIVES 211
METHODS AND MATERIALS 212
PART I: ISOLATION AND ENUMERATION 212
Sample Collection 212
Sample Preparation 212
Selective Media 212
Enumeration 214
Identification 214
PART II: SURVIVAL NETOORK 215
Assembly and use of Chambers 215
Preparation of Pure Cultures for 215
use in Diffusion Chambers
Preparation of Feces for use in 217
Diffusion Chambers
Sanpling of Chambers 217
RESULTS 219
PART I: ISOLATION AND ENUMERATION 219
PART II: SURVIVAL STUDY 219
DISCUSSION 230
PART I: ISOLATION AND ENUMERATION 230
PART II: SURVIVAL STUDY 231
CONCLUSIONS 234
RECOMMENDATIONS 234
APPENDIX 1: BUFFERS AND SOLUTIONS 235
APPENDIX 2: GROWTH MEDIA 236
REFERENCES 238
- 205 -
UST OF TAFtrry;
Table Page Mb.
D-1 Source of envirormiental water used in vitro for the 217
study of bifidobacteria! survival.
D-2 Conparison of levels of bifidobacteria isolated from 220
human feces on YN-17 and MFN.
D-3 Levels of bifidobacteria and E^ coli in human feces 221
as isolated on YN-17 and m-TEC.
D-4 Levels of sorbitol-fermenting bifidobacteria and E^ coli 222
in Lake Ontario water enumerated over a maximum three
day period.
D-5 Mean survival counts of bifidobacteria and E^ coli in 223
Lake Ontario water enumerated over a maximum three
day period.
- 206 -
IiLST OF FIGURES
Figure Page No.
D-1 Dialysis membrane diffusion chamber. 216
D-2 In vitro survival of bifidobacteria isolated from 224
sewage and Ei coli in Lake Ontario water.
D-3 In vitro survial of bifidobacteria isolated from 226
human feces and E^ coli in Lake Ontari water.
D-4 In vitro survival of Bifidobacterium bifidum 227
(ATCC #696) and E^ coli in Lake Ontario water.
D-5 In vitro survival of Bifidobacterium breve 2 2 3
(ATCC #701) and E^ coli in lake Ontario water.
D-6 In vitro survival of fecal bifidobacteria and 229
E. coli in Lake Ontario water.
ATCC
dH20
g
h
HBSA
min
niL
m-TEC
MEN
MRS
YN-17
YII-17(-)
- 207 -
UST OF ABBREVIATICNS
American Type Culture Collection
distilled water
grains
hours
Human Bifid Sorbitol Agar
minutes
milliliters
Medium for selection of thermotolerant E^ coli
Modification of Petuely's PMS for the isolation of
bifidobacteria
Ifedium for the selection of lactobacillia used also for
isolation of bifidobacteria
Medium for the isolation of bifidobacteria
Non-selective medium used to support the growth of
bifidobacteria (Modification of YN-17)
- 208 -
The relatively recent closure of a number of Toronto beaches due to fecal
pollution signifies a need to improve water quality in this area. Outbreaks of
various infections traced to fecal contamination of surface waters are well-
documented (Dart and Stretton, 1980) .
Development of a management strategy plan to reduce fecal contamination
requires suitable detection methods that can locate fecal inputs and
differentiate between human vs. non-human sources of pollution.
Ideally the enumeration of pathogen concentrations in surface waters would
serve to indicate levels of contamination and potential health hazards.
However, pathogens pose problems due to their sporadic occurrence in the
environment and the difficulty in their collection and isolation. Hence, a
more suitable surrogate indicator organism found in feces must be employed.
Geldreich, in 1968, and Bonde, in 1983, have proposed a number of parameters
for an ideal water quality indicator. Briefly stated: i) the indicator should
exist at density levels far exceeding pathogen concentrations in feces of
infected individuals; ii) the indicator should only be present in warm-
blooded animals; iii) there should exist a relatively rapid and inexpensive
means of detecting and enumerating the indicator; iv) indicator's survival
characteristics should parallel those of the pathogen (s) .
The most widely accepted indicator of water quality irrpairment due to human
and animal excrement is the fecal coliform count. The Ontario Ministry of the
Environment utilizes a standard of 100 fecal coliforms/100 mL of water as their
upper limit of safety (MacDonald, 1986) . However, this indicator has a
drawback in that it enumerates Klebsiella which is not restricted to feces
(MacDonald, 1986) .
- 209 -
Escherichia coli is also being utilized as an indicator of fecal
contamination in surface waters. Unlike other fecal coli forms it has not been
isolated from non- fecal sources. Due to its fairly rapid die-off in situ. E.
coli has been enployed as an indicator of recent feccil contamination (Seyfried
et al . , unpublished report) .
Unfortunately, the fecal coliforms, including E^ coli, when used as water
quality indicators, are incapable of distinguishing between human and animal
sources. These organisms are ubiquitously found in higher animals.
Mossel (1958) was the first to propose bifidobacteria as potential
indicators of fecal contamination. Bifidobacterium is a gram-positive,
anaerobic, non-sporulating, non-motile rod often displaying pleomorphic forms
(i.e. Y and V-shaped bifurcated rods) .
Several media have been proposed for the isolation of bifidobacteria. In
1983, Mara and Oragui developed YN-17 medium stating relatively good
selectivity for bifidobacteria. Tanaka and Mutai (1980) stated similar results
with the use of MFN medium, a modification of Petuely's Synthetic Medium (PSM)
(Petuely, 1956). MRS, a medium normally utilized for the isolation of
Lactobacillus spp. has been enployed for the isolation of bifidobacteria by the
Department of Nutrition at the University of Toronto (personal communication) .
Most contamination reported on these three media was the result of fecal
streptococci growth. Lithium chloride was found to control unwanted growth of
Streptococcus spp. . It was found to exert an inhibitory effect at
concentrations of 0.3% and 0.4% in MSI5M Staphylococcus aureus medium (N.A.
KLapes, Annual Meeting, American Society of Microbiologists 1987) . Its
effectiveness in selective media for bifidobacteria has yet to be investigated.
- 210 -
It has been suggested that bifidobacteria could be used to distinguish
between human and animal fecal pollution (Buchanan and Gibbons, 1974; Levin,
1977; Cabelli, 1979; Resnick and Levin, 1981b). However, it was not until 1933
that a method was devised by Mara and Oragui that would allow bifidobacteria to
serve this purpose. Using a sorbitol -based medium (HBSA) , they isolated and
analyzed bifidobacteria from human and animal feces. Their findings showed
that certain sorbitol-fermenting species were restricted to human feces.
The usefulness of bifidobacteria as a specific indicator of human fecal
pollution could be limited due to its short survival in the extra-intestinal
environment (Resnick and Levin, 1981; Oragui, 1982). Survival studies have
shown bifidobacteria to die off more rapidly than E^ coli upon exposure to
environmental waters (Carillo, Estrada and Hazen, 1985) . A too rapid decline
of bifidobacteria in the aquatic environment and subsequent inability to detect
their presence could render the organisms poor indicators of fecal contamination.
In 1972, McFeters and Stuart devised dialysis membrane filter chambers for
in situ and in vitro survival studies. The chamber allows for maximum exchange
of molecules (e.g. gases, organic nutrients, toxic chemicals) between the
environment and a pure culture of bacteria without contaminating the culture by
introducing new water-borne bacteria into it.
Before bifidobacteria can be used as an indicator of recent human fecal
contamination in the Toronto area, their survival in this environment and their
ability to satisfy the parameters of a good indicator must be investigated.
- 211 -
OBJECTIVES OF RESEARCH
1. To assess the suitability of three media: YN-17, MFW, and MRS; in their
selectivity for bifidobac±eria.
2. To enumerate bifidobacteria (all species), bifidobacteria (sorfcitol-
fermenting) and Ej. coli from human feces.
3. To test the inhibitory effects of lithium chloride on bifidobacteria and
fecal streptococci utilizing YN-17.
4. To conpare the survival characteristics, in vitro, of mixed cultures and
laboratory isolates of bifidobacteria with those of E^ coli.
5. To investigate the suitability of bifidobacteria as indicators of recent
human fecal pollution.
- 212 -
ME3IECS AND MftlERIAIS
I^rt 1: Isolatlcai and Enumeratlcai
SAMPLE COLLECYlOtf
Feces were collected from healthy individuals, in sterile, wide-mouthed
glass jars. All sainples were freshly voided and usually examined in the
laboratory within four hours of defecation.
SAMPLE FREPARAnai
A total of 20g of each fecal saiiple was weighted out and transferred to
sterile glass blender jars which had been calibrated previously to a 200niL
volume. Sterile phosphate solution (Appendix 1) was added to the 20CrmL volume
mark producing a 10"^ dilution of the original sample. The diluted samples
were homogenized for 15 seconds at low speed vising a Waring blender.
Systematic 100-fold dilutions were made of the blended samples in sterile 99mL
dilution blanks (Appendix 1) prior to membrane filtering.
Membrane Filtraticn
Dilutions of the fecal samples were membrane filtered according to the
methods outlined in Standard Methods for the Examination of Water and
Wastewater (1980). The filtrations were performed utilizing Gelman 0^6 47ram
cellulose hitrate filters with a porosity of 0.45um. Filters were planted on
the appropriate selective media. For each sample a control was run on the
least selective medium viiich was usually m-TEC.
SEIECnVE MEDIA
m-TEC medium (Dufour, 1975) and the Urease Test (Dufour, 1981) were employed
for the selection and enumeration of E^ coli from the samples tested. The
medium was incubated aerobically for 24h at 44.5° C in the presence of two
plastic containers, holding 50mL of ice each, to ensure a resuscitation step
- 213 -
necessary for target colony growth. The m-TEC medium (Appendix 2) is
selective for all fecal coliforms present in the samples. Target colonies of
all yellow, yellow-green and yellow-brown colonies were enumerated as the fecal
coliform count. To differentiate E^ coli from other thenrotolerant fecal
coliform bacteria, the Urease Test was employed. The sanple filter was removed
from m-TEC and placed onto a sterile filter pad saturated with a urease
solution (Appendix 1) . All urease-negative (yellow) colonies were recorded as
the Ei coli count. .
The MEN medium formulated by Tanaka and Mutai (1980) was utilized to select
for bifidobacteria. MEW medium (Appendix 2) was incubated anaerobically at 37°
C for 48h with GasPaks (BBL) in an atinosphere of 95% hydrogen and 5% carbon
dioxide. Target colonies were round, l-2mm in diameter, convex, flat with an
entire edge and creamy vi*iitish-yellow in appearance.
MRS medium (Appendix 2) was proposed as being selective and specific for
bifidobacteria (Department of Nutrition, University of Toronto, 1987) . The
medium was incubated anaerobically at 37° C for 24h with GasPaks (BBL) in an
atmosphere of 95% hydrogen and 5% carbon dioxide. Presumptively positive
bifidobacteria colonies were those that were circular, 2-3ram in diameter, dark
green or blue, convex, and mucoid. Fecal streptococci contamination was
observed as the presence of colonies l-2mm in diameter round and green with a
pale green periphery.
The YN-17 medium (Appendix 2) formulated by Mara and Oragui (1983) was also
utilized for its selectivity and specificity for bifidobacteria. The medium
was incubated anaerobically for 48h employing the same anaerobic techniques
used for MRJ and MRS media. Target colonies and background contamination were
identified by the same criteria used for the MRS medium.
- 214 -
Human Bifid Sorbitol Agar (HBSA) (Appendix 2) was formulated by Mara and
Oragui (1983) . The medium was used to select for sorbitol-fermenting
bifidobacteria. Incubation procedures were consistent with those used for YN-
17 medium. Target colonies were taken as those colonies that were deep yellow
and brownish-yellow, 2-3ram in diameter, circular and convex. Contamination of
fecal streptococci were those colonies measuring l-2mm in diameter, flat, pale
yellow or colorless.
Concentrations of 3% and 4% LiCl were incorporated into a separate batch of
YN-17 media to test for suppression of fecal streptococcal growth. Incubation
procedures and target and non-target identification criteria were consistent
with those utilized for the YN-17 medium.
ENUMERATICN OF BTFTDOBACTERIA AND E. CPU
Human fecal sanples were obtained and processed as mentioned above.
Enumeration of bifidobacteria and E^ coli was performed using the membrane
filtration method and the two media, m-TEC and YN-17.
JEEtTCIFlCfinCtl OF BIFIDOBACrERIA
Colonies from YN-17, ME^, and MRS were picked and streaked on YN-17 (-)
(Appendix 2) to purify. Isolates were classified as gram-positive or gram-
negative organisms. Gram-positive rods exhibiting the typical pleomorphic
morphology were restreaked and incubated in aerobic and anaerobic environments.
Those isolates growing anaerobically were presumed to be bifidobacteria and
were subjected to further biochemical tests. The confirmation was performed by
Inja Huh (1986-87) using the following biochemical tests: catalase, gelatin,
arginine, bile esculin, and KLiger's iron. Further identification of the
isolates to the species levels was accomplished using carbohydrate fermentation
tests (Huh, 1986-87) .
- 215 -
Part U: Survival Study
ASSIMBKir AND USE OF DIFHJSiai OffiMEERS
SOmL dialysis mentorane diffusion chambers used in the survival study were
designed by McFeters and Stuart (1972) . The chamber consists of a three-part
plexiglass II cell with membrane filter sidewalls (Fig. 1) . Filter sidewalls
were made of a polycarbonate film with a 99mm diameter and a porosity of 0.2um
(Nucleopore) . Filters were placed between the plexiglass outer walls and the
centre piece. Wing nutbolts were loosely set in place to hold the assembled
chambers together. The chambers were autoclaved for 25min. at 121° C (ISlbs
pressure) . Before use, the wing nutbolts were tightened.
E5?EPARATICN OF KIKE BACTERIAL CUIIIURES PCR USE IN DIFFUSiaJ CHAMBERS
Pure cultures of bifidobacteria and E^ coli were prepared by inoculating 5mL
of MRS broth (Appendix 2) with a single colony from a pure culture of bacterium
grown overnight on YN-17(-) (Appendix 2). The broth cultures were incubated
for 48h at 37° C. Broth cultures were subjected to centrifugation at 300 rpm
for 10 min. The supernatant was discarded and the pellet resuspended in 5mL of
chilled gelatin phosphate buffer (Appendix 1) and recentrifuged. This step was
repeated twice. After the supernatant was discarded, the cells were
resuspended once again in 5mL of sterile gelatin phosphate buffer and measured
spectrophotometrically at 660nm. For bifidobacteria an optical density reading
of greater than 1.0 signified a culture content of approximately 10^ bacterial
cells. For E^. coli. an optical density reading of greater than 1.1 signified a
culture content of approximately 10^ bacterial cells. Further preparation of
the cultures was performed as follows:
- 216
Figure D-1
DIALYSIS DIrFUSIOri CHAiOZR
Ji~
y
r-
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(McFeters i Stuart, 1.
- 217 -
i) Tenfold dilutions of the culture were made up to a 1 in 10^ dilution of the
original culture in 99inL dilution blanks (Appendix 1) . The dilutions were
analyzed by meinbrane filtration and the filters placed onto appropriate
selective media (i.e. m-TEC for E^ coli and YN-17 for bifidobacteria) . The
media were incubated according to the mEdium and genus requirements, as
earlier stated, and subsequent colony counts were obtained to calculate the
cell density of the original pure culture.
ii) A 1 in lO'hiiL dilution was used to fill the chaii±>ers so that the
approximate cell density would fall between 10'^ and 10^ organisms per
mL. This dilution was achieved by transferring 3mL of the 1 in lO^mL
dilution to a flask containing 297mL of sterilized environmental water.
Table 1 presents a list of the locations from which the environmental
water was obtained for each culture utilized in the survival study.
The sterile, assembled chambers were inoculated with the pure bacterial
culture using a sterile 50mL luer-lock syringe. The chambers could
accommodate slightly more than the specified volume and were therefore
filled to capacity. The chambers were then placed in a glass tank
containing environmental water at room temperature.
FREPARATiai OF FECES FOR USE IN DIFFUSION CHAMBERS
A human fecal sample was obtained and prepared as outlined in Part I,
Materials and Methods, of this report. The sample was diluted to a 1 in 10%L
dilution and 3mL of this dilution were placed in 297mL of sterilized
environmental water. Chambers were filled as stated above.
Table 1
' SOURCE OF ENVIRONMENIAL WATER USED IN VITRO
FOR THE STUDY OF BIFIDOBACTERIAL SURVIVAL
ENVIRONMENTAL WATER
LOCATION
lake Ontario: Harbourfront
Queen City Yacht Club dock
Lake Ontario: at foot of Brimley Road
Lake Ontario: Kew Beach near Woodbine
Race Track
IDENl'lFICATTON
TYPE OF ISOLAT]
CODE
308
fecal isolate
701
B. breve
696
B. bifidum
P-Z
feces
EY-6
sewage isolate
- 218 -
SAMPUNG OF CHAMBERS
The chambers were sanpled at 2, 5, 12, 24, 48, and 72 hoiars using lOmL
disposable luer-lock syringes. The syringe was locked onto the port possessing
the attached capillary tube and pumped five times to resuspend settled cells
prior to removing the sample. Volumes from 1 to 12mL were removed,
appropriately diluted, membrane filtered, and planted on both YN-17 and m-TEC.
Bacterial densities were enumerated after incubation.
NOTE: All samples for the survival study were performed in triplicate.
- 219 -
RESunrs
Part 1: Isolatlcn and Enumeratlcn
Of the three media tested for their selectivity and specificity for
bifidobacteria from human feces, MRS displayed a high density of contamination.
Colonies obtained included streptococci, and lactobaccili as well as
bifidobacteria. Hence, the medium was found to be non-specific for
bifidobacteria .
YN-17 and MFN were found to be equally selective for bifidobacteria with
less than 3% contamination by fecal streptococci. Table 2 shows that the two
media were equal in their ability to recover bifidobacteria from the fecal
samples.
MFN required an incubation time of 48h before visible growth occurred,
exceeding the incubation tinie required for YN-17 by 24h.
Results of enumeration of bifidobacteria on YN-17 and E^ coli on m-TEC are
presented in Table 3. Bifidobacterial counts from human feces were found to be
greater than counts of E^ coli by a factor of approximately 10-^. Table 4
presents results obtained using HBSA. Sorbitol-fermenting bifidobacteria were
found in equal densities to those of E^ coli. YN-17 medium supplemented with
LiCl concentrations of 3% and 4% displayed fecal streptococci contamination
accounting for < 8% of the total colonies isolated.
Part 2: Suivival of Bifidohacteria and E. ooli (Table 5)
Pure Culture of Bifidobacteria Isolated frcm Sewage
The die-off patterns of bifidobacteria and E^ coli appeared to parallel each
other (Fig. 2) . However, bifidohsacteria could not be recovered after the
- 220 -
TABLE D2
COMPARISON OF LEVELS OF BIFIDOBACTERLA
ISOLATED FROM HUMAN FECES ON YN-17 AND MPN
Bifidobacteria Per 1 Gram
SAMPLE of Human Feces
SOURCE
YN-17 MPN
Human I 2.1 x 10^ 2.0 x 10^
Human A 1.8x10^ 2.0x10^
Mean 2.0 x 10^ 2.0 x 10^
=LE D3
- 221 -
LEVELS OF BIFIDOBACTERIA AND ESCHERICHIA COLI
IN HUMAN FECES AS ISOLATED ON YN-17 AND M-TEC
SAMPLE
SOURCE
Microorganisms Per Gram of Feces
Bifidobacterium spp
Escherichia coli
Human A
Human B
Human C
Human D
Human E
Human F
Human G
Human H
2.3 X 10'
2.6 X 10'
1.1 X 10'
1.2 X 10
7.9 X 10
4.1 X 10
10
10
10
1.4 X 10
11
1.8 X 10
10
1.8 X 10
2.0 X 10
1.6 X 10
9.4 X 10
5.0 X 10
1.6x 10
3.4 X 10
2.5 X 10
Mean
3.7 X 10
10
2.4 X 10
enumerated on YN-17
enumerated on M-Tec
r.^31E Cii
_ ")2'' -
LEVELS OF SORBITOL-FERMENTING BIFIDOBACTERIA
AND ESCHERICfflA COU IN HUMAN FECES AS
ISOLATED ON HBSA AND M-TEC
SAMPLE
SOURCE
Microorganisms Per Gram of Feces
Bifidobacterium spp
Escherichia coli
.b
Human A
Human B
Human C
Human D
Human E
Human F
Human G
Human H
2.3 X 10'
1.2 X lo'''
3.2 X 10^
4.0 X 10^
2.2 X 10^
4.2 X 10''
1.9 X 10^
3.0 X lO''
1.8 X 10'
2.0 X 10'
1.6x 10
9.4 X 10
5.0 X 10
1.6 X 10
3.4 X 10'
2.5 X 10
Mean
2.0 X 10'
2.3 X 10'
enumerated on HBSA
enumerated on m-Tec
- 11
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- 225 -
second day of exposure. E^ coli, in ODntrast, was able to survive as long as
48h after ejqxDsure to the environmental water, but was not detectable after the
third day. After the 15h mark the bifidobacteria appeared to decline somewhat
more rapidly than E^ coli.
Pure Culture of Bifidobacteria Isolated frcm Human Feces
The bifidobacteria declined more quicJcly than E^ coli within the first 2h of
exposure with the bifidobacterial cultures decreasing greater than one order of
magnitude and the E^ coli cultures decreasing less than one order of magnitude
(Fig. 3) . Bifidobacterium cultures died off within 24h of exposure to the lake
water in vitro.
Pure Culture of Bifidobacterium bifidum (ATOC #696)
The longevity of Bi f idobacter ium bifidum was relatively consistent with tliat
observed with the E^ coli culture. However, this pattern appeared to change
after 5h where the B^ bifidum decreased in numbers much faster than E^ coli.
After 24h, B^ bifidum could only be isolated from one chamber in low numbers
(i.e. < l/lmL) . (Fig. 4) .
Pure Culture of Bifidobacterium breve (ATOC #701)
Bifidobacterium breve presented a more rapid decrease in density for the
most part of the survival analysis when compared to E^ coli. Decline of
B. breve was more marked after 5h of exposure and the species could not be
recovered after 24h. (Fig. 5) .
Fecal Sanple
Both Bifidobacteria and E^ coli appeared to survive maintaining fairly
steady densities between 0 and 12h (Fig. 5) . However, rapid decline of the
bifidobacteria occurred after this time interval whereas E^ coli seemed to
decrease in numbers much more slowly.
- 225 -
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- 227 -
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- 230 -
DISOJSSICN
Part 1: Isolation and Enumeraticn
One of the major problems in assessing an organism's potential role as a
water quality indicator stems from difficulties in its isolation. Therefore a
coirparison of the three media: YN-17, MRS, and MPti for their selectability and
specificity was performed.
Although visible target colonies were observable on MRS after a relatively
short incubation time (i.e. 24h) , the medium was not found to be sufficiently
specific for bifidobacteria. The MRS medium was prepared utilizing the
antibiotic cocktail devised for YN-17 (Appendix 2) . However, this formulation
was not able to suppress the occurrence of excessive contamination by
lactobacilli and fecal streptococci. This occurrence is best explained by the
fact that the MRS medium was richer in nutrient content than the other two
media, having been originally developed to support the growth of
Lactobacillus spp. (Dept. of Nutrition, University of Toronto, personal
communication) . Although MRS was not appropriate for the specific isolation of
bifidobacteria, it was utilized (void of antibiotics) , in broth tubes to
support the growth of pure cultures of these organisms. MRS, as a nutrient
medium, was found to be suitable for bifidobacteria with visible growth
appearing as early as 16h after incubation.
YN-17 and MFN appeared to be equally selective for bifidobacteria as seen in
Table 2. The results stated previously show that the two media were also
comparatively specific for bifidobacteria v^ere background fecal streptococci
were found in levels no higher than 5% on either media. However, as it was
earlier stated, a potential water quality indicator should possess a relatively
rapid means of detection and enumeration (Bonde, 1978) . In this regard, YN-17
- 231 -
was superior to MPN in that YN-17 required a 24h shorter incubation period in
comparison to MFN.
Fecal streptococci were found to be the most successful competitor to
bifidobacteria where on occasion, the former would overgrow the latter on YN-
17. Hence a means to better suppress the grcwth of fecal streptococci on this
medium was sought. Attempts to suppress fecal streptococci contamination using
0.3% and 0.4% LiCl in YN-17 failed. Fecal streptococci contamination prevailed
and were found in numbers consistent with those obtained prior to the
incorporation of LiCl.
The use of E^ coli as a water quality indicator of recent fecal
contamination (Seyfried et al. . unpublished report) provides a standard with
which to compare bifidobacteria. For the reasons mentioned above, YN-17 was
the mediijm of choice for the enumeration of bifidobacteria from feces and
subsequent density level comparisons with E^ coli. Also employed was Mara and
Oragui's (1983) HBSA medium for the enumeration of sortoitol-fermenting
bifidobacteria.
The mean density levels of bifidobacteria and E^ coli found in the feces of
eight human adults have been tabulated (Table 3) . Similarly, the density
levels of sorbitol-fermenting bifidobacteria in comparison with E^ coli have
been presented in Table 4. The results of this study showed bifidobacteria to
exist in feces at a 1000-fold higher concentration than E^ coli. The sorbitol-
fermenting species of bifidobacteria presented themselves in numbers coitparable
to those of Ei coli (i.e. 10^) in the sample tested. These values were
consistent with the bacterial content, found in human feces, reported by Mara
and Oragui (1983). i
- 232 -
Part 2: Survival Study
Figures 2-6 represent the results obtained form the survival study. In
every case both bifidobacteria and E^ coli were found to decline after
exposure to Lake Ontario water, in vitro. Possible reasons for the observed
die-off include: nutrient depletion, diffusion of nutrients out of the dialysis
chambers, toxic chemical diffusion into the chamber, and where bifidobacteria
is concerned, the effects of atmospheric oxygen levels toxic to the anaerobes.
Pure cultures of bifidobacteria appeared to parallel the die-off trends in
Ei coli up to the 5h mark; thereafter bifidobacteria began to show a slightly
more rapid decline (Figures 2, 4, 5). In all cases bifidobacteria died off
within 24h of exposure whereas E. coli persisted as long as 48h.
As was noted, the bifidobacteria survived was comparable with E^ coli within
the first 5h of exposure. It is suggested that, upon exposure to the
environment, some of the bifidobacteria were able to utilize nutrients from
dead cells to maintain their existence. Eventually as time passed the effects
of diffusion, resulting in dilution of nutrients and ejqx)sure of the pure
culture to any toxic chemicals present in the water, may have had an inpact on
the survival of the organisms. Also, it is expected that competition for
nutrients increased over time due to the depletion of nutrients by the
organisms themselves. The more rapid decline of bifidobacteria in comparison
to Ei coli may possibly be accounted for by considering oxygen diffusion into
the chamber. Initially the pure cultures were suspended in autoclaved
environmental water (see Methods and Materials) . Heating of the water would
have led to a subsequent driving-off of oxygen. Once the chambers containing
the suspension of bifidobacteria were placed into the lake water, oxygen
content may have increased due to diffusion. Thus it was perhaps the toxic
- 233 -
levels of oxygen after the 5h mark that lead to the more rapid decline of the
bifidobacteria at this point.
Figure 3 presents bifidobacteria and E^ coli in fairly steady decline with
both cultures becoming undetectable within 24h. The more rapid decline of both
organisms in comparison to those cultures presented in Figures 2, 4 and 5, may
have been the result of differences in environmental water utilized in the
study. Table 1 shows that the water used for the cultures (Fig. 3) was
retrieved from a boat dock. It is quite possible that this water contained
residual chemicals, emitted by boat engines, that would have a toxic effect on
the bacterial cultures.
Results of exposure of a mixed culture of bifidobacteria are presented in
Table 6. In this instance, a diluted fecal sample was introduced into the
dialysis chamber in an effort to present bifidobacteria and E^ coli in vitro,
as they might be found in the natural environment.
As can be seen in Figure 5, both organisms tended to exist in a relatively
stationary state over a 12h period. These results are inconsistent with the
survival data found using the pure cultures. However, it would be plausible to
state that the greater nutrient level provided by the diluted fecal material
within the chamber, allowed the organisms to maintain their numbers.
It is possible that coitpetition with other organisms found in human feces,
nutrient depletion, and effects of toxic chemicals resulted in the decline of
the bifidobacteria and E^ coli after 12h exposure to the environment.
Once again the more rapid decline phase of bifidobacteria is suggested to
have been due to the fact that the organisms are strict anaerobes.
- 234 -
OCNCmSICNS
Coiiparisons with E^ coli, presently being used as a water quality indicator
of recent fecal pollution (Seyfried et al. , unpublished report) , support the
assumption that sorbitol-fermenting species of bifidobacteria may serve as
indicators of recent fecal contamination in surface waters. As was shown in
this study, their densities in feces, their ability to survive in vitro for as
long as 24 hours, and the existence of rtvethods allowing for their selection and
recovery si:ipport this proclamation.
Unfortunately an in vitro survival study poses some limitations. The
effects of ultraviolet radiation, temperature changes, drying, predation,
competition, water flow, and pH changes occurring in the natural environment
could never be mimicked completely in vitro.
Nonetheless, the fact that certain species of bifidobacteria have been found
to be restricted to human feces, and their tendency to die off quite rapidly
make these organisms promising water quality indicators for determining
pollution sources and locations.
REOCMMENEftnCNS
1. A study should be performed in Ontario surface waters to characterize the
survival trends of bifidobacteria in situ.
2. An epidemiological study should be performed in Ontario to confirm the
statements that sorbitol-fermenting species of bifidobacteria are restricted
to humans.
- 235 -
APraMDZX 1
Riosphate Soluticn/Diluticn Blanks:
a) Dissolve 34. Og KH2PO4 in SOOmL dH20. Adjust pH to 7.2. Dilute to IL
with dH2) .
b) Dissolve 50g MgS04 in IL dH20.
Magnesium Chloride Soluticn:
MgCl2 38. Og
Distilled water (dH20) l.OL
Stir ingredients to dissolve. Autoclave for 15min. at 121° C (15 lbs
pressure) .
Diluticai Blanks:
Phosphate Solution (1) 1.25mL
MgCl Solution (2) 5.0inL
Mix solutions and dispense 99mL (plus 4inL for evaporation) into dilution
blanks. Autoclave for 15min. at 121° C (ISlbs pressure) .
Gelatin Riosphate Buffer:
gelatin 2g
Na2HP04 3.7g
NaH2P04 7 . 25g
dH20 l.OL
Dissolve in SOOmL of distilled water. Adjust pH to 7.2 ±0.2 with l.ON
NaOH. Dissolve gelatin in SOOmL of distilled water (heat to < 90° C) .
Autoclave for ISmin. at 121° C (llSlbs pressure) .
Urease Reagent:
Urea 10 . Og
Ehenol Red O.OSg
Ethanol 0 . 5mL
Mix ingredients and adjust pH to 5.0+0.2.
- 236 -
AEFENDIX 2
Growth Media
1. MRS (oxoid) broth was prepared according to the manufacturer's
recornmendations .
2. MRS selective medium was prepared by addition of th YN-17 antibiotic
cocktail (4a) and 20g of BactoAgar.
ra-TEC Agar:
Proteose peptone #3
S.OOg
Ye^st extract
3.00g
Lactose
lO.OOg
NaCl
7.50g
K2HPO4
3.3g
KH2PO4
l.OOg
Sodium lauryl sulphate
0.20g
Sodium deoxycholate
O.lOg
Bromocresol purple
0.08g
Bromocresol red
0.08g
Agar
IS.OOg
dH-.0
l.OOL
Mix above ingredients and heat to 90° C to dissolve. Autoclave for 15min.
at 121° C (151bs pressure) . Cool to 50° C and dispense in sterile petri
dishes . Final pH 7 . 1+0 . 1 .
4a. YN-17
Yeast extract
20. Og
Polypeptone (BBL)
10. Og
Lactose
10. Og
Casamino acids
8.0g
NaCl
3.2g
Ecxjmocresol green
0.3g
Cysteine hydrochloride
0.4g
BactoAgar
15. Og
dH20
IL
Antibiotics:
Nalidixic acid
0.03g
Kanamycin sulphate
0.05g
Polymixin B
0.006g
Mix above ingredients with the exception of the antibiotics and heat to
90° C to dissolve. Autoclave for 15min. at 121° C (151bs pressure) . Cool
to 50° C and add antibiotics listed above. Dispense in sterile petri
dishes. Final pH 6.9+0.2.
b. YN-17 (-):
Same formulation as above with the exclusion of antibiotics.
5. Human Bifid Sorbitol Agar (HBSA) :
- 237 -
Sorbitol
10. Og
Polypeptone (BBL)
10. Og
Yea.st extract
20. Og
Casamino acids
8.0g
NaCl
3.2g
Bronocresol purple
O.lg
Cysteine hydrochloride
0.4g
Bacto Agar
15. Og
dH20 l.OL
Antibiotics:
Nalidixic acid
0.03g
Kanamycin sulphate
0.05g
Polymixin B
0.0012g
Mix above ingredients with the exception of the antibiotics and hat to 90°
C to dissolve. Autoclave for 15min. at 121° C (151bs pressure) . Cool to
50° C and add antibiotics listed above. Final pH 6.8+0.2.
MFN:
a)
b)
Lactose
20. Og
(NH4)2S04
5.0g
K2HKJ4
l.Og
Twef^n 80
l.Og
Bromocresol
purple
o.ooieg
Bacto Agar
20. Og
Salt Solution:
(S.OmL)
FeS04*7H20
0.5g
MnS04*2H20
0.4g
MgS04*7H20
10. Og
NaCl
0.3g
dH20
250mL
Biotin
O.OOOlg
Pantothenic
acid
0.002g
Riboflavin
O.OOlg
Adenine
O.OOlg
Guanine
O.OOlg
Xanthine
O.OOlg
Uracil
O.OOlg
Mix ingredients (a) in 740inL dH20 and heat to 90° C to dissolve.
Autoclave for 15min. at 121° C (151bs pressure) . Cool to 50° C. Mix
ingredients (b) in lOmL dH20 and sterilize by meiitjrane filtration. Add
(b) to (a) and adjust pH to 6.8+0.2.
- 238 -
REFEEENCES
1. Bonds, G.J. (1977) Bacterial indicator of water pollution. Adv. Aquatic
Microbiol. I: 273-364.
2. Buchanan, R.E. and Gibbons, N.E. (1974) Bergey's Manual of Determinative
Bacteriology ei^th edn. Williams and WilJcins: Baltiinore.
3. Cabelli, V.J. (1979) Evaluation of recreational water quality: the EPA
approach. in BioloqiccLL Indicators of Water Quality. A. James and L.
Evison (ed.), John Wiley and Sons: Oiichester. pp. 1-23.
4. Carillo, M. , Estrada, E. and Hazen, T.C. (1985) Survival and enumeration
of the fecal indicators Bifidobacterium adolescentis and Escherichia coli
in a tropical rain forest watershed. Amer. Soc. for Microbiol. 5.0(2) :
468-476.
5. ■ Dufour, A.P. and Cabelli, V.J. (1975) A membrane filter procedure for
enumerating the conponent genera of the coliform group in seawater. Appl.
Microbiol- 29: 826-833.
6. Dufour, A.P,, Strickland, E.R. and Cabelli, V.J. (1981) Membrane filter
method for enumerating Escherichia coli. Appli. and Environ. Micrcbiol.
4195): 1152-1158.
7. Geldreich, E.E., Best, L.C., Kenner, B.A. and VonDonsel (1968) The
bacteriological aspects of stormwater pollution. J. WPCF. 40(11): 1861-
1872.
8. Huh, I. (1986-87) The isolation and identification of Bifidobacteria.
University of Toronto, unpublished report.
9. Levin, M.A. (1977) Bifidobacterium as water quality indicators. in
Bacterial Indicators/Health Hazards Associated with Water. W.W. Hoadley
and B.J. Dutka (ed.). ASTM Publications: RvLladelphia . pp. 131-138.
10. MacDonald, J. (1986) Humber River bacterial sources and pathways study.
Technical Rf^xDrt #13. A report of the Toronto Area Watershed Management
Strategy Steering Committee. Prepared for the Ontario Ministry of the
Environment.
11. Mara, D.D. and Oragui, J.I. (1983) Sorbitol-fermenting bifidobacteria as
specific indicators of human faecal pollution. J. Appl. Bact. 55: 349-
357.
12. Mossel, D.A.A. (1958) The suitability of bifidobacteria as part of a
more extended bacterial association indicating fecal contamination of
foods. Seventh Intematicnal Ocngress of Microbiology Abstracts of
Papers. Almquist and Wikesells: Uppsala, pp. 440-441.
13. McFeters, G.A. and Stuart (1972) Survival of fecal coliform bacteria in
natural waters. Field studies with membrane filter chambers. App.
Micrcbiol. 24: 805-311.
- 239 -
14. Oragui, J.I. (1982) Bacteriological methods for the distinction between
human and animal fecal pollution. Hi.D. Thesis, University of Cundee,
Scotland.
15. Petuely, F. (1956) Ein einfacher vollsynthetischer Selektiv nahrboden fur
den Lactobacillus bifidus. Zentralbl. Bakeriol . Prasitenkd.
Infekticnskr. I^. Abt. I. Orig. 166: 95-99.
16. Resnick, K.G. and Levin, M.A. (1981) Assessment of bifidobacteria as
indicators of human fecal pollution. Appli. and Evotcti. Micrcibiol- 42:
433-438.
17. Seyfried, P et al. (1987) unpublished report.
18. American Public Health Assoc. (1985) .qharrtaTri Methods for the
T=^fnmirwl-icn of Water and Wastewater. 16th edn. AFHA, AWWA and WPCF (ed.).
pp. 886-901.
19. Tanaka, R. and Mutai, M. (1980) Iitproved medium for selective isolation
and eniimeration of bifidobacterium. Appl. and Envircn. Micrc±)iol. 40:
866-869.
- 240 -
STECIAL NOTES
There seenved to exist sane discrepancies between the findings of this
study and those of Mara and Oragui (1983) in regards to YN-17 medium. Mara
and Oragui found flat, non-ntucoid colonies measuring 2-4iTim and dark green in
colour to be background streptococci. Ihey distinguished domed, mucoid
colonies measuring l-2mm and pale green with a pale green periphery as being
bifidobacteria.
However, this study showed the opposite to be true. This is to say the
smellier dark green/blue flatter colonies were identified as bifidobacteria and
those ejdiibiting a pale green periphery were contaminating streptococci or more
likely a mixture of bifidobacteria and streptococci. Any colonies pale green
in colour that did not contain a darker centre were found to be streptococci.
- 241 -
APPDJDIX E
1
Clostridiun perfrinqens and Bifidobacterium ^. as Tracers in Storm Sewers
Eric K. Hani,
Department of Microbiology
Iftiiversity of Torcnto
- 242 -
TAHTF OF acwTENrs
Page No.
LIST OF TABLES ^^^
LIST OF FIGURES ^^^
245
METHODS
Sample Collection ^^-^
245
Analysis
253
SUMMARY '^
- 243 -
UBT OF TS^HLES
Table Page No.
E-1 Ratio of Ei coli to Clostridiuni perfrinqens 246
and of Bifidobacteria to Ej_ coli for High
Priority and Non-Priority Storm and
Sanitary Sewage
E-2 Geometric Mean Concentrations of Fecal 247
Coliforms, E^ coli, Clostridium perfrinqens
and Bifidobacteria in Hi(^ Priority,
Non-Priority Storm and Sanitary Sewage
- 244 - ,
UST OF FIGURES
Figure Page No.
E-1 Geometric Mean Concentrations of Fecal 248
Coliforms, E^ coli. Bifidobacteria spp. and
Clostridium perfringens in Sanitary Sewage
during Dry Weather Survey June 10, 11
and 12, 1987
E-2 Geometric Mean Concentrations of Fecal 249
Coliforms, E^. coli. Bifidobacteria spp. and
Clostridium perfringens in High Priority
Storm Sewage during Dry Weather Survey
June 10, 11 and 12, 1987
E-3 A conparison of the Geometric Mean 250
Concentrations of E^ coli, Clostridium
perfringens and Bifidobacteria in Hit^
Priority and Non-Priority Storm and
Sanitary Sewage (DOG values)
E-4 A comparison of the Geometric Mean 251
Concentrations of E^ coli, Clostridium
perfringens and Bifidobacteria in Human,
Cat and Dog Feces (LOG values)
E-5 Percentage of Sorbitol Fermenting 252
Bifidobacteria in Feces - High Priority
and Non-Priority Storm and Sanitary Sewage
- 245 -
METHODS
Sanple Collecticn
1. High priority storm sewage- triplicate sanples from three in-line sample
points.
2. Sanitary sewage-triplicate saitples fron three in-line saitple points.
3. Non-priority storm sewage-triplicate saitples from two in-line sample
points.
4. Fecal samples from humans, cats, dogs.
Analysis
Membrane filtration for parameters:
Fecal coliforms/E. coli
m-TEC + urease treatment
44.5 ± 0.5° C; 23 + 1 hr
(Dufour, Strickland and Cabelli, 1975)
Clostridium perfringens
Heat treatment at 70° C for 30 minutes
mCP-2 at 37° C for 48 hr
(Ontario Ministry of the Environment, 1986)
Bifidcbacteriim sp.
YN17 at 37° C for 48 hr, anaerobic gas pack, anaerobic jar
(Mara and Oragni, 1983)
isolates tested in sorbitol broth)
- 246 -
Table E-1
Ratio of E. Ooli to Clostriditnn perfrinqens and of Bif idcbacteria to
E- CDli for Hi^ Priarity and Non-Priority Stonn and Sanitary Sewage
Sewer Sanple
HP Storm A
HP Storm B
HP Storm
HP Storm
NP Storm
NP Storm
Sanitary
Sanitary
Sanitary
48
141
5.
7.
7.
83
50
150
E/C = Ei Coli; Clostridium perfringens
Seuec Sanple
B/E
HP Storm A
1.7
HP Storm B
2.3
HP Storm Y
1.9
HP Storm C
0.25
NP Storm X
0.01
NP Storm Z
0.04
Sanitary D
0.02
Sanitary E
0.02
Sanitary F
0.05
B/E = Bifidobact
:erium/E. Coli
- 247
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- 253 -
SUMMARY
Clostridium perfrinqens
present in feces in hi^ density
gcxxi survival ciiaracteristics
no other source in the environment
expensive to recover
no human/non-human determination possible
indication of age of pollution
indication of water treatment performance
Bifidobacterium spp.
indication of recent pollution
human/non-human determination possible
no other source in the environment
present in feces in high concentration
no replication extraintestinally
easy to identify
- 254 -
AFFQnnx F
CHRRftCIHaZftTICN OF PEEDEOCNAS AOHGINDSA
FRO* STC»M AND SANITRRY SEWERS
KCm. HABMANDAYAN
EEPARIMEOT OF MICRDBIOIJDGY,
UNTVERSnY OF "KMINID
- 255 -
TABI£ OF OCNIOnS
Page No.
I. INTRDCUCnON 258
II. OBJECTIVES OF RESEARCH 260
III. MMERIAIS AND METHODS 260
Sanple Collection 260
Enumeration of Pollution Indicator Organisms 260
Isolation of Pseudomonas aeruginosa 261
Biochemical Tests 261
Serotyping 264
Genotyping (REA) 264
IV. RESULTS
Enuineration of Fecal Indicator Bacteria in Sewers. 266
Storm Sewers 266
Sanitary Sewers 268
Serotyping 268
Genotyping . 272
V. DISCUSSION 276
Enumeration of Fecal Indicator Bacteria
in Sewers 276
Serotyping 273
Genotyping 283
VI. CONCUJSION ■ 286
VII. APPENDIX 288
Buffers and Solutions . 288
Growth Media 289
VIII. BIBLIOGRAPHY 292
- 256 -
UST OF IS^ELES
Table ' Page No.
F-1 Media and Incubation Parameters for Enumerating Selected 262
Bacterial Grotps
F-2 Serotype Distribution of Pseudomonas aeruginosa in Storm 271
and Sanitary Sewage
F-3 Result of Agarose Gel Electrojiioresis of Total Cellular DMA 274
Extracted From Pseudomonas aeruginosa
F-4 Correlation of Various Serogroup Typing Schemes Based on 0 Ag 280
F-5 Worldwide Frequency of Incidence of Serotypes of 282
Pseudomonas aeruginosa
- 257 -
LIST OF FIGURES
Figure Page No.
F-1 Gec3metric mean cxjncentration of fecal indicators 267
in storm sewage
F-2 Geometric mean concentrations of fecal indicators 269
in sanitary sewage
F-3 Percentage serotypes of Pseudomonas aeruginosa 270
in storm and sanitary sewage
F-4 Agarose gel electrophoresis of total cellular DNA 273
from Pseudomonas aeruginosa digested with Sma I
endonuclease
F-5 Agaixjse gel electrophoresis of total cellular DNA 275
from Pseudomonas aeruginosa digested with SMa I
endonuclease
F-6 Agarose gel electrophoresis of total cellular CNA 277
from Pseudomonas aeruginosa digested with SMa I
endonuclease
F-7 Agarose gel electrophoresis of total cellular DNA 234
from Pseudomonas aeruginosa digested with six
different endonucleases
- 258 -
Incnr^ased concern for public hecilth hazard has lead to methods aimed at
reducing the input of fecal pollution in surface water bodies. Human fecal
material in sanitary sewage, contains bacteria which may cause infections in
the appropriate host. Under normal circumstances, this sanitary sewage is
treated in wastewater treatment plants, reducing bacterial numbers, and the
effluent is discharged into natural waterways. This process serves to mininiize
the contact between humans and human fecal material thereby decreasing the
probability of infection.
Recently, the Toronto Area Watershed Management (TAKM) has shown that
storm sewers contribute to the fecal pollution of the Humber and Don Rivers.
Originally these sewers were designed for channelling storm water from urban
areas into surface waters to avoid the flooding that would have resulted, as a
consequence of the bloc3djig of natural flow patterns that existed before
urbanization. The bacterial content of storm sewers should be similar to
direct runoff and have virtually no human fecal irpjt.
High fecal bacterial levels detected in storm sewers suggests that these
sewers may be contaminated with human fecal wastes. Ihe problems resulting
from this are obvious: if sewage enters the surface water supplies untreated,
the probability of infecting a suitable host is high. Therefore, in order to
determine which home connections are contributing to fecal pollution, some
method of tracing the pollution must be developed.
One opportunistic pathogen that has been recovered from human fecal
material and sewage is Pseudomonas aeruginosa. Ringen and Drake (1952) have
shown that even though Pseudomonas aeruginosa can be isolated from a wide
variety of natural sources, its distribution is limited. They found that
- 259 -
Pseudomonas aeruginosa was not isolated in natural waters of mountain streams
or deep wells remote from human habitation and free of human waste material.
In addition Wheater and coworicers (1978) have shown that Pseudomonas aeruginosa
was not found in a variety of animal feces. The presence of this organism in
barnyards, animal feces and soil was due to chance and close association with
man. Therefore, the presence of Pseudomonas aeruginosa in surface waters and
storm sewage may indicate the presence of human sanitary wastes.
Subspeciation of Pseudomonas aeruginosa has classified this microorganism
into 17 different serotypes according to their somatic-O-antigen. Studies by
Seyfried and Fraser (1977) and Young and Moody (1974) have shown that tracing
the Pseudomonas aeruginosa infection in clinical and environmental settings is
possible. Therefore, by adopting their methods of pollution source tracings in
storm sewers, one may be able to determine the presence of human sanitary waste
in storm water and trace the location of the inputs.
With a limited amount of information on the distribution of serotypes of
Pseudomonas aeruginosa and their distribution in the environment, work must be
performed to determine if there is a difference between serotypes of
Pseudomonas aeruginosa found in humans and those from environmental sources.
If a serotype, specific to sanitary wastes, if found, then isolating the same
serotype from storm sewers might indicate the presence of sanitary waste.
Another method vAiich may prove valuable in determining if a specific type
of Pseudomonas aeruginosa is associated with human feces, is restriction enzyme
analysis. This method may be able to determine if a specific Pseudomonas
aeruginosa genotype is present in human fecal material as compared to
environmental sources. This in turn may serve as the probe used to determine
the presence of human Pseudomonas aeruginosa contamination in storm sewers.
- 260 -
OBJECTIVES OF RESEARCH ■:
The objectives of this research were to:
1. Characterize isolates of Pseudomonas aeruginosa from sanitary and storm
sewage according to their serotypes and genotypes; in order to see vvtiat
differences and/or similarities exist among isolates obtained from these
types of waste and to apply this knowledge to develop a method of detecting
the presence of sanitary waste in storm sewage.
2. Etetennine which method (serotyping or genotyping) will give more discrimina-
ting information about a specific strain of Pseudomonas aeruginosa .
MATERIALS AND MEIHCCS
3.1 Sanple Collection
Sairples were collected from three different points along the storm sewer
line, in triplicate, over a three day period.
Samples were also collected at three points along the sanitary sewer
lines.
Water samples were collected in sterile Sodium Thiosulphate EDTA treated
bottles (Appendix) and transported to the laboratory on ice. Upon receipt of
the samples, analysis was performed within three hours.
3.2 Enumeration of Pollution Indicator Organisms
Appropriate dilutions, ranging from 50ml to lO"'^, of each water sample
were made in phosphate solution (Appendix) . Water samples were analyzed using
standard membrane filtration technigues (APHA, 1989) . Water samples were
filtered through 0.45 ^^m porosity membranes (Gelman Sciences) and
- 261 -
subsequently placed on selective media. Table 1 lists the media, incubation
times and temperatures used for quantitation of different bacterial groups.
3.3 Isolatioi of PseudamoTas aenninosa
Approximately 10 bacterial colonies were picked form m-PA plates, after
the incubation period, and streaked onto nutrient agar (Difco) plates for
isolated colonies. Plates were incubated at 35° C for 24 hours. One isolated
colony was picked from the nutrient agar plates and incubated at 35° C for 24
hours. Cultures were maintained on BHI (Difco) slants overlaid with sterile
paraffin oil. These slants were maintained at 20° C.
3.4 Bioctiesnical Tests Performed en PseiidnnnrBS aeruginosa
An inoculum of the culture was removed from the EHI slant and streaked on
BHI agar plates for two consecutive transfers. Plates were incubated at 35' C
for 24 hours.
1. - 3% KDH Test
The 3% KDH test (Fluharty and Packard, 1967) was used to determine the
gram reaction of each isolate. A small inoculum of the culture was mixed
with a drop of 3% KDH solution on a slide. The suspension was observed
for consistency.
Gram-positive organisms did not gel when mixed with KOH while gram-
negative organisms (Pseudomonas aeruginosa) form a thick stringy gel.
Results were recorded immediately.
- 262 -
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- 263 -
2. - Oxidase Production
A 1% solution of N, N, N, N- tetramethyl -p-phenylene diamine (Appendix)
was used to detect the presence of oxidase. The solution was impregnated
on clean Whatman No. 5 filter paper discs and allowed to dry. Cultures
were taken from heart infusion agar plates with sterile toothpicks and
rubbed into the treated filter disc.
Cultures possessing oxidase produce a purple colour on the filter paper,
while the negative cultures remained colourless. Results were recorded
within two minutes, Pseudomonas aeruginosa is oxidase positive.
3. - Acetamide Reaction
Each Pseudomonas isolate was streaked onto acetamide agar slants
(Appendix) and incubated for 24 to 36 hours at 37° C. The production of
a dark pink (purple) coloration of the slant by Pseudomonas aeruginosa
was regarded a positive result.
4. - Skim Milk Reaction: (Brown and Foster, 1970)
Skim Milk agar (Appendix) was used to determine four reactions; pigment
production, casein digestion, fluorescence and grape-like odour by
Pseudomonas aeruginosa . Pigment was read as green, yellow or brown.
Casein digestion was evidenced by production of a clear zone in the agar
around each isolated colony. Cultures were also tested for their ability
to fluoresce under a Wood's lamp. Pseudomonas aeruginosa cultures were
found to digest casein, produce a grape-like aroma, have a green-yellow
pigment, and fluoresce blue-green under UV light.
- 264 -
3.5 - Serotyping
Serotyping Kit (Difco number 3081-32) was used to serotype Pseudomonas
aeruginosa. Before serotyping, the diluted antisera was tested against its
specific antigen (Difco number 3082-32) for positive control, also it was
tested against rabbit serum for negative control.
A day before serotyping each pure Pseudomonas aeruginosa was streaked out
on nutrient agar and incubated at 37° C for 18 to 24 hours.
The day of serotyping, the culture was swabbed with a sterile swab, and
suspended in 0.85% saline. The suspension was autoclaved for 30 minutes at
121° C, followed by centrifugation at 1000-2000 rpm for 10 minutes. The
si^jematant was discarded and the bacterial mass was resuspended in 0.75 ml
Merthiolate saline (1:10000 Merthiolate in 0.85% NaCl solution) .
This constitutes the stable heated antigen and it was tested against each
antisera on slide agglutination.
3.6 Restriction Enzyme Analysis of PsFaximiCTTas aeruginosa
Total cellular DMA was extracted as follows: (Bradbury et al, 1984;
Bradbury et al, 1985) .
A 1.5ml volume of an 18h nutrient broth culture inoculated with
Pseudomonas aeruginosa was transferred into a 1.5ml Eppendorff tube and
centrifuged in a Microfuge 12, Bec3<man for three minutes at 7500 x g. The
supernatant was discarded and the pellet loosened by vortexing. A 291 ul
volume of FEB I buffer (Appendix) containing lOmg/ml lysozyme was added and the
mixture incubated for 20 minutes at 35° C. A 9ul volume of 5M NaCl was added
and mixed well. A 150ul volume of 10% SCS (Appendix) was added. The solution
was mixed gently and incubated for ten minutes at 37° C. After 450 >ul volume
- 265 -
of 25:24:l:phenol: chloroform: Isoamyl was added to the tube, the mixture was
vortexed and centrifuged for six minutes at roan temperature at 7500 x g. The
upper aqueous phase was removed with a pasteur pipet and transferred into a
l.Sral Eppendorf tube. One ml of 95% cold ethanol was added, the tubes
vigorously shaken and stored at -20° C overnight. The mixture was centrifuged
for three minutes at 12000 x g, the supernatant discarded and the pellet
redissolved in 250 ml Dt^ wash buffer (Appendix) . A total of 500 ul of 95%
cold ethanol was added, the mixture stored at -20° C for 20 minutes, and
centrifuged at 1200 x g for three minutes. Ihe supernatant was discarded and
the pellet allowed to dry at 37° C for 10 minutes. Ihe pellet was dissolved in
100 ul of distilled water and stored at 4° C until digested.
Restriction digests were performed using Sma I according to the
manufacturer's instructions (Boehringer Mannheim) . A 10 ul aliquot of 2x Sma I
buffer was delivered to an Eppendorf tube and 10 ul of extracted n^ was added.
A 2 ul sartple of Sma I enzyme was added to the mixture and incubated for one
hour at 37° C for conplete digestion to occur. A one ml volume of 0.15M CDIA +
0.4 mg/ml RNase A was added and incubated for 20 minutes at 37° C. A five ul
volume of 5x sairple buffer was added to the restriction digest. Samples were
electrophoresed on 0.7% agarose gel for 16 hours at 27 volts. Gels were
stained with one mg/ml Ethidium bromide in 1 x TAE (Tris base, 1.0 sodium
acetate, 0.1 M disodium EDIA) for one hour and destained for 3 hours in
distilled water. Photography was done using UV light at 300nm and a red No.23A
polaroid 665p/N film with an exposure time of 30 seconds.
- 266 -
Results
4.1 Enumeraticn of Fecal Indicator Bacteria in Sesirers
Fecal coliform, E^ coll. fecal Streptococci and Pseudonionas aeruginosa
density were performed on all sanples from storm and sanitary sewers, using the
membrane filtration technique and media described in Table 1.
4.1.1 Storm Sewar
- Fecal coliform count in storm sewer varied from 10-^ - 10** coliform per
100ml (Fig. 1) .
Ei coli counts were in the 10-^ E^ coli per 100ml range (Fig. 1) .
- The level of streptococci was relatively consistent at three points.
- Pseudomonas aeruginosa levels were lew cotrpared to other bacterial
indicators. Storm sewers should not contain any human fecal iiput, and
therefore should have low numbers of Pseudomonas aeruginosa organisms.
Geldreich and Kenner (1969) proposed that the sources of fecal bacterial
pollution may be differentiated by using a fecal coliform to fecal streptococci
FC/FS ratio. It was suggested that if FC/FS ratio is greater than 4.0, the
source of pollution is likely of human origin; while if the ratio is less than
0.7 then the source is probably of non-human origin, FC/FS ratios between 0.7
and 4.0 have been considered as intermediate mixed. FC/FS ratios must be
eirployed with some degree of caution as it is time dependent; once discharged
into receiving waters, the differential dieoff rates of these organisms and
diverse environmental factors may alter their interrelationship to such an
extent as to render the FC/FS ratio of limited or no significance in
determining the source of bacterial contamination.
- 26-
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- 268 -
The three sites contained very high counts of fecal col i forms. Usually,
storm sewers contain little feccLL contamination, however at this time it is not
known v*iat kind of contamination is contributing to the counts observed.
4.1.1 Sanitary Sewer
Sanitary sewers usually contain human fecal waste material ^ich is
treated before being deposited in water bodies.
Fecal coliform, E^ coli. feccil streptococci concentrations were almost 10
fold more than the levels observed in storm sewers (Fig. 2) .
The Pseudomonas aeruginosa count was observed to be around 10000 per
100ml in the sanitary sewers. This was considerably hi^er than the counts
observed in storm sewers.
4.2 Serotyping
In total 285 isolates were serotyped; 148 isolates from storm sewers and
137 isolates from sanitary sewers. The distribution of Pseudomonas aeruginosa
serotypes in storm and sanitary sewage varied (Fig. 3 and Table 2) .
In storm sewers (Table 2), the most prevalent serotype was 0:6, about
72.3%; followed by serotypes 0:1(15.5%), 0:16(5.4%), 0:11(4.7%), 0:8(1.1%) and
0:12(0.7%).
Sanitary sewers also had serotype 0:6 as the predominant serotype
(56.2%). Other serotypes observed from sanitary sewers were serotype
0:10(21.9%), 0:11(13.1%), 0:1(4.4%) and serotype 0:2, 0:4, 0:5, 0:7 were
present in very low levels (1.5%) .
Serotype 0:16 only appeared in storm sewers, v^ereas serotype 0:10 was
found only in sanitary sewers; however, the percentage recovered was not as
high as serotype 0:6.
- 269 -
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- 271 -
Taole F-2
Total nuncar of isolataa : 2S5
Stom sswqr
Sanitazry a ewer
1-3
137
Nimbsr 01" isola-^s 39ro~v:D=c
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- 272 -
4.3 Genotyping
Genotyping was carried out according to the Bradbury et al. 1984,
Bradbury et al. 1985 method. Isolates randomly chosen with different serotypes
were subjected to restriction enzyme analysis.
Figure 4: -
Lane 1 contained lambda bacteriophage marker digested
with Hind III.
Lane 2-8 contained isolates from storm sewers with
serotype 0:5.
Lane 9 and 10 contained isolates from sanitary sewers
with serotype 0:6.
Examining Figure 4, it is possible that the same serotype from storm or
sanitary sewers have different genotypes (restriction pattern) .
Lanes 2, 3, 4, 7 and 8 have the same restriction pattern whereas lanes 5
and 6 have different banding patterns from the previous ones.
Lanes 9 and 10 are from sanitary sewers, serotype 0:6, but each has a
different restriction pattern compared to the other isolates.
Four different patterns with genotype 0:6 were observed (Fig. 4, Table
3) : Two different patterns from storm and two different patterns from sanitary
sewers.
Figure 5:
lanes 2 to 6 contain isolates from sanitary sewers with
serotype 0:6.
Lanes 7 and 8 contain isolates from sanitary sewers
with serotype 0:1.
Lanes 9 and 10 contain isolates from storm severs with
serotype 0:1 Not all isolates with serotype 0:6 had
the same genotype (Fig. 5), lanes 2, 3, 5 and 6 were
identical to each other, whereas lane 4 had a totally
different banding pattern.
_ T7-) _
Lan9
Source
Serotyp*
RZA Pattam
Figure F-4
1
2
3
i\
5
6
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9
10
2
s
6
s
6
3
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3
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A
A
A
B
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A
A
C
D
Agaro3e (0.7,S) gal el9ctrophor«3is of to^tal
cellular DMA from PS^UDOITONAS AZ;iUGINOSA
digeatsd with Sma I andonuclaaaa.
s : atorm
St sanitary
- 274 -
Table ?-3
5LZ3UL- C? AG^JIGSZ (C?.^) '3ZL ZlZCTRCrHCHZSIS CJ
DNA 3L':TR.\CTi:D FHCM PSZUDOI.IONAS .^RUGIZtOSA R-^'iDOr.!,'/ :
JX'J
ISCLATS NuT.IBZR SII£ SEROTYPE 1--.::Z RZ^ FATTEr.:;
PXG '4 332 storm A 6 2
^00 storr: 563
^20 storr. 36^ A
1^55 stom A 6 7 ---
1^90 zzoirz 3 6 S
T'Z S 1235 sani^sr;/ ? 5 2
13^5 3arJ.-ary ? 6 2-
13^0 ss-ii-ary Z c 5
1570 sarJ-tary Z 6 5 .-
— T^ u o"! ' o — -,v~- J. i< r -
rij H :^— ^ o«a-.u .- ^ ^ —
9^0 sxorr: 3 6 6 ' 2
53s sajiitary F 6 9 C
1505 sanitar:.- D 6 10 D
I52B sanitazy D 6 iJ- Z
-I527
1530
^{-21
i^36
FIG 6 361
393
sanitary
sanitary
D
D
1
1
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callular DNA from PSETOOHONAS AZHUGOOSA
digaatad with ima I andonuclaase.
s « «torm
3 : sanitary
- 276 -
Lanes 7 and 8 had the same restriction pattern and both
were from sanitary sewers serotype 0:1; however, when
these were coirpared to lane 9 and 10 they displayed a
different restriction pattern. Serotype 0:1 from
sanitary sewer had a different fingerprint from storm
sewer isolates with serotype 0:1.
Coitparing Figure 4 to Figure 5, seme serotype 0:6
organisms from storm sewer had an identical pattern
or serotype (Table 3) , to sanitary sewer serotype
0:6. Therefore, in this case, serotype 0:6 from
storm sewer had two different genotypes whereas the
sanitary sewer had four different genotypes.
Figure 6
Lanes 2 and 3 had isolates from storm severs with
serotype 0:16; both had identical patterns whereas
lanes 4 and 5 contained isolates from sanitary sewer
serotype 0:10 with totally different genotype each.
Lane 6 contained an isolate fron sanitary sewer with
serotype 0:11 which had different pattern compared to
serotype 0:11 in lanes 8 and 9 from storm sewers.
Lanes 7 and 10 each contained different serotypes, 0:4
from sanitary sewer and 0:8 from storm sewer, they
had totally distinct restriction pattern from each
other.
DISCUSSICN
5.1 Enumsratlcn of Fecal Indicator Bacteria
The result of the study (Fig. 1 and Fig. 2) show that Pseudomonas
aeruginosa was found in high concentrations in the human sanitary sewers in
relation to high fecal indicator counts. Pseudomonas aeruginosa was also
recovered in the storm sewers although in Icwer concentration than in the
sanitary sewer. The concentration in storm sewer of both fecal col i form and E.
coli was high especially at point A and approached levels of 10,000
organisms/ 100ml. FC and EC concentration of this magnitude are suspiciously
high for storm water and suggest the presence of sanitary contamination or an
extra input from somewhere else. At this point conclusions can not he irade
about the source of the contamination.
- 277 -
Lana
Source
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Agarose (.0.7ji>) gel elsctrophorssis of tota]
cellular DNA from ?S£U0CKONAS AERUGINOSA
digaatid with S.Tia I ^ndonuclsaaa .
s : jtorm
3 t sanitary
- 278 -
5.2 Serotyping
The cxsncept of serological heterogeneity of Pseudonpnas aeruginosa was
first recognized in 1912 by Jacob Sthal, who reported that the 55 strains he
was investigating were not serologically hcmogenecus (Verdes and Evans, 1961) ,
The heterogeneous nature of this bacterium was also noted in 1916 by
Trommsdorff , who emphasized the iirportance of distinguishing between the
different types of Pseudomonas aeruginosa (Verder & Evans, 19161) . These
authors failed, however, to recognize and differentiate between the heat stable
(0) and heat labile (H) antigenic ccaiponents of Pseudomonas aeruginosa. The
presence of two different antigens was first r^xDrted by Brutsaert (1924) and
the different antigenic conponents were investigated specially (Monoz et al.
1945, 1949; Gaby, 1946; Mayr-Harting, 1948; Fox and Loutoury, 1953; L^wbury and
Fox, 1954) ; however, the develcpnent of a practical typing scheme, based on the
antigens of Pseudomonas aeruginosa, was slow.
Habs in 1957 used boiled antigens and immune sera prepared against these
suspensions to devise the first antigenic scheme, suitable for the practical
differentiation of Pseudcanonas aeruginosa. She reported the differentiation of
70 strains of Pseudomonas aeruginosa into 12 serogroups, based on heat stable 0
agglutinogens. Cross reactions between grtxps 0:2 and 0:5 were evident and
there appeared to be a common antigen among all the strains. Kleinmaier (1957)
confirmed Habs' work using slide agglutination with living suspensions of
Pseudomonas aeruginosa.
SandviJc (1960) developed an antigenic scheme for this bacterium
containing seven 0 groups, into which he could subdivide 87 strains of
Pseudomonas aeruginosa of animal origin. When it was compared with Habs' 6/7
serogroups were the same, but the uncommon serogroup was added to the Habs'
scheme as 0:13. Veron (1961) analyzed Habs' cross reacting serogroups 0:2 and
- 279 -
0:5 and subdivided 0:2 into 0:2a and 0:2b and 0:5 into 0:5c and 0:5d. Veron
also emphasized the necessity of cross absorption in order to eliminate the
cross reactivity and produce monospecific typing serum thus Veron described 10
O-groups and 10 H-grot^s.
lanyi (1966/67) further extended the work of Verder & Evans (1961) ; he
reported 13 0 types of whicii 5 would be further divided.
Fisher et al. (1969) described a new concept in serotyping of Pseudonxpnas
aeruginosa. Tliey differentiated strains of this bacterium on the basis of
protective antigens and reported on immunotypes.
Homma et al. (1970) developed a typing scheme, based on her 0-serotypes,
tested by tube agglutination.
Bergan (1973) coipared the typing sets of Habs, Lanyi and Sandvik and
reported as the others had, that they were similar. Since then an
international panel under the auspices of the subcommittee on Pseudomonas
aeruginosa and related organisms came v^) with 17 different serotypes.
Types 1 -12 were pr^ared using Habs' culture
Type 13 with Veron 0:13 (Sandvik's type 11)
Type 14 with Verder and Evans' 5
Type 15 with Lanyi 's 12
Type 16 with Homma 's 13
Type 17 with Meitert's type X strain
Table 4 shows the correlation of various serogroups typing schemes based
on 0 antigens up to 1969.
Serotyping of Pseudomonas aeruginosa was carried out using "Pseudomonas
Antisera" Kit (Difco) which utilizes the heat stable 0-antigen. Of the 285
isolates serotyped, 148 were isolated from storm sewers, and the remaining 137
from sanitary sewers.
280 -
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Table 2 lists the distribution of serotypes of Pseudomonas aeruginosa in
storm and sanitary sewage. Serotype 0:6 was the most frequently identified
serotype in storm and sanitary sewers, 72.3% and 56.2% respectively. Lanyi
(1966, 1967) has also r^xsrted that the same serotype was the most common
serotype isolated from sewage and surface waters.
Serotypes 0:1 and 0:11 were commonly isolated from storm and sanitary
sewers (Table 2) . These serotypes were also frequently encountered in studies
by Muraschi and coworkers (1966) , Lanyi (1966/67) ; Table 5 shows worldwide
frequency.
Serotype 0:16 was only recovered from storm sewers, with a frequency of
5.4% and serotype 0:10 recovered frcxn sanitary sewers 21.9%. This suggests
that serotypes 0:16 and 0:10 each belong to a specific source. In theory,
storm sewers should only have direct run off from streets, domestic animals,
and birds' fecal contamination and sanitary sewers should have human waste
inputs. Therefore, since type 0:10 was found in a high frequency in sanitary
sewers, and was absent from storm sewers, this serotype may be specific and
indicate human fecal contamination. However, at the present time it is not
known if serotype 0:10 is specific to humans. Sampling of fecal material from
domestic and wild animals should be performed in order to determine if serotype
0:10 is found in animal wastes and if so, at v^^iat frequency?
If serotype 0;10 was found to be specific to human feces, water samples
of storm sewers taJcen from various points could be performed and tested for the
presence of Pseudomonas aeruginosa serotype 0:10. If the sample was positive
for serotype 10, then the sampling site could be examined and the source of the
illegal connection determined.
Serotyping by itself does not differentiate within a specific serotype.
E'/en though serotypes 0:1, 0:6 and 0:11 were common to both storm and sanitary
- 282 -
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sewers, this does not mean that they are identical. To ascertain if this was
the case, restriction enzyme analysis was performed on serotype groups in order
to determine if identical genotype patterns are present within each serotype
groi^j.
5.3 Genotyping (REA)
Initially REA was performed on three isolates using six different
restriction enzymes. Ihis step was used to determine which restriction enzyme
cut the genomic CNA and produced the best banding patterns on polyacrylamide
gels. Figure 7 displays the different banding patterns that were obtained.
Sma I produced the best pattern because hi(^ molecular weights of CNA bands
were distinct and separate unlike the bands produced by Hind III, Bam Hi, Eco
RI, KFN I and Xho I. Furthermore Sma I is six base cutter cuts at GGGCCC
points since Pseudomonas aeruginosa has high G/C content of 67%. This
indicates that the hi<^er G/C content of Pseudomonas aeruginosa may make it
more susceptible to cutting by Sma I.
Twenty-seven isolates of Pseudcanonas aeruginosa were chosen from storm
and sanitary sewage and genotyped. Isolates were chosen randomly according to
their serotype.
Figure 4 displays the genotypes of sewer isolates from storm sewers,
serotype 0:6 and two isolates from sanitary sewers, serotype 0:6. The banding
pattern of the storm sewer isolates Figure 4 lanes 5 and 6 were not consistent
with others. Isolates in lanes, 2, 3, 4 and 5 are identical where those in
lanes 9 and 10 differ. In comparison, five isolates from sanitary sewers,
serotype 0:6 Figure 5, lanes 2 -6 did not display the same genotype pattern.
Isolates in lanes 2, 3, 5 and 6 were identical, while the isolate in lane 4 was
different.
- 234 -
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- 285 -
Identical genotype patterns were observed using serotype 0:16 (Figure 6
lanes 2 and 3). Serotype 0:1 fron storm Figure 5, lanes 9 and 10 were
identical whereas serotype 0:1 from sanitary Figure 5 lanes 7 and 3 were
identical but different frcm storm.
Different genotype patterns were observed within serotype 0:11 from storm
sewer Figure 6 lanes 8 and 9 and serotype 0;10 Figure 6 lanes 4 and 5.
Differences within the specific genotype were noted in the high molecular
weight positions, usually at MW 23.13 K daltons.
Differences were also noted when coraparing serotype 0:6 sanitary Figure 4
lanes 9 and 10, and serotype 0:6 storm Figure 4 lanes 2-8.
Comparing Figure 4 and Figure 5, it is possible to conclude that sane
serotypes 6 from storm and sanitciry sewer are identical (i.e. they have the
same genotype Table 3) , but at this tiite one cannot make any conclusion about
the presence and the source of this genotype in storm sewers.
The differences that were seen in REA were not observed using the
serotyping technique. For example, not all serotype 0:6 isolates possessed the
same genome pattern. This may be accounted for by the fact that the genes
responsible for serotyping the 0-Ag canprise a small percentage of the total
genome. Therefore, when comparing total chromosamcLL CNA other genes besides
those that code for the 0-Ag, which is characteristic of serotype
differentiation, will appecir. Thus, the technique of REA proves to be highly
specific, in that it further subdivides one serotype into a branch of different
genotypes whic±i can then be used in tracing bacteriail contaminants from
sanitary sewers into storm sewers.
It should be noted that genotypes of Pseudomonas aeruginosa isolates have
not been reported by other researchers.
- 286 -
Serotyping, used by itself, was insufficiently discriminatory and of
little ^idemiological value as some serotypes, namely 0:1, 0:6 and 0:11 were
cammonly encountered. Conversely, genotyping produced highly specific bands
and differences within strains of Pseudcnonag aeruginosa were readily
demonstrated.
It has been previously suggested that serotype 0:10 '^as e^/ident only in
sanitary sewers. In addition, the genotype banding patterns were observed to
be different (Figure 5 lanes 4 and 5). If serotype 0:10 was found in low
frequencies in other animal groups, REA may provide highly discriminatory
genotypes specific to humans that can be used in tracing the source of input of
Pseudomonas aeruginosa in storm sewers Iron a specific site.
Future studies using a probe developed from either total chramosomal CNA
or a specific fragment common to human strains will allow us to hybridize
against cLLl other strains.
Ihis information would prove valuable in differentiating between human
and non-human fecal waste.
(XNCmSICN
Some common serotypes of Pseudomonas aeruginosa are present in both
sanitary and storm sewers. The most frequently occurring serotype was 0:6.
Serotype 0:10 appears to exist only in sanitary sewers whereas serotype
0:16 was found only in storm sewage. However no definite conclusion can be
made at this time as to the origin of these serotypes (i.e. sanitary or non-
sanitary contamination) without further study of the Pseudomonas aer'jginosa
serotypes found in human and animal feces and those recovered from non-fecally
polluted environments.
- 287 -
Genotyping is more discriminative tJian serotyping, since it can highlight
both differences and similarities within a given serotype.
This suggests that REA could be used to specially identify strains from
sanitary wastes in storm sewage.
- 288 -
APPEMUX
7.1 Buffers and Soluticns
1. Sodivm Ttiinpailphate/UJIA:
^2^2*^3 " ^2^ 2-^*?
EDIA 37 . 2g
Distilled water (dH20) 100. 0ml
Stir ingredients to dissolve. Add 0.3ml of buffer to each 120ml sample
bottle. Autoclave 20 min. at 121° C (15 lbs. pressure) .
2. Efiosphate Soluticxi:
(A) Dissolve 34. Og KH2PO4 in 500ml dH20.
Adjust pH to 7.2.
Dilute to 1 liter with dH20.
(B) Dissolve 50g y!gSO^ . 7H2O in IL dH20.
Autoclave both solutions sepeirately for 15 minutes at 121° C. Cool ard
store at 4° C for up to 1 month. Add 1.25ml of (A) ard 5ml of (B) to IL
of dH20. Dispense as dilution blanks or for rinse water. Autoclave for
20 minutes at 121° C. (15 lbs. pressure) .
3. Saline (0.85%)
Add 8.5g of NaCl per litre of dH20. Dispense 5ml in each test tube.
Autoclave 15 minutes at 121° C. Store at 4° C.
4. FEB I
SOnM Glucose, lOmM (EDIA), 25iiiM Tris HCL (FH 8.0)
lOmg/ml lysozyme
5. CNA Wash Buffer
0.1 M sodium acetate, 50mM ^DPS FH(8.0)
6. 5 X Sample Buffer
1.0ml of 50 X TAE
2.5ml of 1% Brtarcphenol Blue in 50% ethanol
7. 50 X TAE
302. 5g Tris base, 136. 08g sodium acetate
tri-hydrate (or 82.03g of anhidrous)
37. 5g Na2EEnA . 2H2O
- 289 -
7.2 Growth Media
The following media (Difco) was pr^ared according to manufacturer's
reccamnendation :
1. ' Nutrient agar 1.5%
2. Nutrient broth
3. EHI agar
4. Skim MiUc Agar:
SJdjn miUc power lOOg
Agcu: 15g
dH20 LL
Add skim milk powder to 500ml dH20. Stir without heat for 30
minutes. Add agar to 500ml dH2 and heat to dissolve.
Autoclave solutions separately for 12 minutes at 121' C. Cool solutions
to 55' C. Add milk to agar solution aseptically. Mix thoroughly and
dispense into plates. Final pH 6.4 +/- 0-2 . Store at 4H C.
5. Medium for the Isolaticn of ttv^ttto Tolerant E. ooli
(m-TEC - Mug agar) :
Proteose peptone No. 3 10. Og
Yeast extract 4.0g
Lactose 5 . Og
NaCl 7.5g
KH2HPO4 3 . 3g
KH2PO4 l.Og
Sodium Lauryl Sulphate 0.2g
Sodium deoxycholate O.lg
Agar 15. Og
MDG 0.05g.
( 4-inethylumbellif eryl-B-D-glucuronide)
dH20 IL
Mix above ingredients except MDG to IL dH20 and heat to 90' C to
dissolve. Add MUG just beforn autoclaving. Autoclave for 15 minutes
at 121' C. Cool to 55-60' C and dispense in sterile square petri
dishes. FL'-al pH 7.1 */- 0.1. Store at 4' C.
- 290 -
Agar (mENT) :
M-Enterococcus agar (Difco) 42g
Sterile dH20 IL
Wei(^t out agar in a sterile beaker using an alcohol flamed spatula.
Heat to dissolve agar (93° C) . Cool and dispense into square petri
dishes. Final pH 7.2 +/- 0.2.
Medium for Pspiiimmas aeruginosa (m-PA) :
L-lysine monohydrochoride 5.0g
Yeast extract 2.0g
Xylose 2.5g
Sodium thiosulfate 5.0g
Magnesium Sulphate, anhydrous 1.5g
Sucrose 1.25g
lactose 1.25g
Sodium C2iLoride 5.0g
Ferri ammonium citrate 0.30g
Sodium disoxycholate O.lOg
Hienol red O.OSg
dH20 sterile 800ml
Mix above ingredients and adjxjst pH to 7.6. Add 15g agar. Heat to
93° C to dissolve agar. Cool to 60° C and stir in Antibiotic
solution*. Dispense into square sterile petri dishes. Store at 4° C
after solidification. Final pH 7.1 +/- 0.1.
* Antibiotic solution:
A) Sulfapyridine 0.1760g
B) Kanamycin sulphate 0.0085g
C) Naladixic acid 0.0370g
D) Cycloheximide 0 . 1500g
Dissolve A-D in 200ml sterile dH20. Heat to 50° C to dissolve
antibiotics.
- 291 -
Acetamids Agar Slants
NaCl 5g
K2HPO4 1.4g
KH2PO4 0.7g
Acetamide 10. Og
I^ SO4-7 H2O 1-Og
Ehenol red 0.012g
Agar 15. Og
dH20 U;.
Add all ingredients except agar. Mjx:st pH to 6.8 then add agar.
Heat to 92' C, autoclave for 15 minutes at 121' C. Final pH 6.3 V
0.2. Dispense into sterile screw Ccip test tubes.
- 292 -
RTnr.TOGRftFHY
Berrgan, T. 1973. Epidemiology Mar]cers for Pseudcmonas aerugincsa . 1.
Serotyping, Pyocine Typing and their Interrelations. Acta Pathol.
Microbiol. Scand. 81: 70-80.
Bergan, T. 1973. Epidemiology Markers for Pseudcmonas aeruginosa. 2.
Relationships between Bacteriophage Siosceptibility and Serogroup and
Pyocine Type. Acta Pathol. Micrcbiol. Scand. 81: 81-90.
Bergan, T. 1973. Eipidemiological Markers for Pseudoroonas aeraginosa. 3.
Comparison of Bacteriophage Typing, Serotyping, and Pyocine Typing on a
heterogeneous Clinical Material. Acta Pathol. Microbiol. Scand. 81: 91-
101-
Bradbury, W.C. , A.D. Pearson, M.A. Marto, R.V. Congi and J.L. Penner. 1984.
Investigation of a Campylobacter jejuni outbreak by serotyping and
chromosamal restriction endonuclease analysis. J. Clin. Microbiol. 19:
342-346.
Bradbury, W.C, R.G.E. Murray, C. Mancini and V.L. Morris. 1985. Bacterial
chramosomal restriction endonuclease ancilysis of the homology of
Bacteroides species. J. Clin. Microbiol. 21: 24-28.
Brown, M.R.V. and Foski, J.H. 1970. A simple diagnostic milk medium for P.
aeruginosa. I of clinical pathology. 23: 172-177.
Brutsaert, P. 1924. L'antigene des Baciles Pyocyaniques . C.R. Soc. Biol.
Paris, 90: 1290-1292.
Fisher, M.W. , Devlin, H.B. , and Qiabasik, F.J. 1969. New Immunotype Schema for
Pseudomonas aeruginosa Based on Protective Antigens. J, Bact. 98: 835-
836.
Fluharty, D.M. and Packard, W.L. 1967. Differentiation of Gram Positive and
Gram Negative Bacteria Without Staining. Amer. J. Vet. Clin. Pathol. 1:
31-35.
Galbraith, J.H. and Williams, R.E. 1972. Migration and Leaching of Metals from
Old Mine Tailings Deposits. Groundwater 10: 33.
Habs, I. 1967. Untersuchungen Uber Die 0-Antigene von Pseudomonas aeruginosa.
Z. Hyg. 144: 218-228.
Homma, J. Yuzuru, K.S. Kim, H. Yamada, M. Ito, H. Shionoya, and Y. Kawabe.
1970. Serological Typing of Pseudomonas aeruginosa and its Cross
Infection. Jap. J. Exp. Med. 40: 347-359.
Kleinmaier, H. 1957. Die O-Gruppenbestinroung von Pseudomonas - Stammen Mittals
objekttrager - Agglutination. Zbl. Bakl., 170: 570-583.
Lanyi, B. 1966/67. Serological Properties of Pseudomonas aeruginosa. 1. Group
Specific Somatic Antigens. Acta Microbiol. Acad. Sci. Hung. 13: 295-313.
- 293 -
Lanyi, B. Gregacs, M. , and Adams, M.M. 1966/67. Incidence of ?seudciTK:nas
aeruqinosa Serogroups in Water and Human Feces. Acta Microbiol. Acad.
Sci. Hung, 13: 319-326.
Moraschi, T. , Bolles, D.M. , Moczulske, C, and Lindsay, M. 1966. Serological
Types of Pseudononas aeruginosa Basfid on Heat Stable 0 Antigens:
Correlation of Habs' (European) and Verder and Evans' (North American)
Classifications. J. Inf. Dis. 116: 84-88.
Ringen, L.M. and Drake, C.H. 1952. A StLKly of the Incidence of Psei^omcnas
aeruginosa frcan Various Natural Sources. J. Bact. 64: 841-845.
Sandvik, 0. 1960. Serological Caiparison Between Strains of Pseudcincnas
aeruginosa from Human and Animal Sources. Acta Pathol. Microbiol. Scard.
48: 56-60.
Seyfried, L-P. and Eraser, D.J. 1978. Pseudomonas aeruginosa in Swimming Pools
Related to the Incidence of Otitis Externa Infection. H.L.S. 1591) : 50-
57.
Verder, E. and Evans, J. 1961. A Proposed Antigenic Schema for the
Identification of Strains of Pseudanoncts aeruginosa. J. Inf. Dis. 109:
133-193.
Veron, M. 1961. Sur 1 ' agglutination de Pseudomonas aeruginosa: Subdivison des
Groupes Antigeniques 0:2 et 0:5. Annales Inst. Pasteur, 101: 456-460.
Wheater, D.W.F., Mara, D.D. , luzan Jawad and Oragui, J. Biological Lndicatcrs
of Waste Quality A. James and L. Evison, (et) John Wiley and Sons (Pub) .
1979. Ql. 21.
Young, V.M. and Moody, M.R. 1974. Serotyping of Pseudomonas aeruginosa. J.
Inf. Dis. J. 130 (S): S47-S51.
- 294 -
AHBOnX - G
mi'lDOBfi^'IT^l? 1 1 JM SP.
Bifidobacteria are gram-positive organisms which may have club-shaped,
bifid ends. Colonies are about lnm in diameter, smooth, entire, convex,
opaque, glistening and white. Bifidobacteria found in huinans include 3^
adolescentis . B. catenulatum. B. breve, B. lonqum. B. globosum and B. infant is.
Even though they have been isolated, their significance has not been determined
because biochemical fermentation reactions, and microscopic examination do not
definitely differentiate between all Bifidobacterium species. Polyacrylamide
gel electrophoresis of cellular proteins or other genetic investigations, which
can give conclusive identification, can be time-consuming.
The only pathogenic Bifidobacterium sp. is B. dentium which is found in
the mouth, intestines, and in mixed infections of the Icwer respiratory tract.
Because it is an obligate anaerobe, it produces acetic and lactic acids in
peptone-yeast-extract-glucose broth and ferments many sugars. According to GIC
results, bifidobacteria produce more acetic in comparison with lactic acid
than other gram-positive, non-sporeforming anaerobic bacilli in humans.
Bifidobacteria recovery from membrane filtration is hampered by the growth
of other bacteria on the media. In YN17 (Mara and Oragui, 1983) , a media used
for bifidobacteria cultivation, the antibiotics present prevent growth of other
bacteria facilitating isolation and making identification easier. However, the
addition of actidione (cycloheximide) to inhibit mold growth, and the addition
of specific vitamins, minerails or other antibiotics, have been considered to
fortify the media.
- 295 -
Bifidobacteria have t±ie ability to alter their appearance and this makes
their classification difficult.
Msfthcd of Recaverlna isoi^t-iac!
Bifidobacteria isolates were obtained by diluting a sample and passing it
through a membrane filter.
On YN17 (blue) , bifidobacteria appears as very dark blue to black
colonies. The green-tinged colonies are usually streptococci. Other media
used included MRS (de Man et al. 1960) by Gibco or Oxoid. It was suggested
that by the addition of cysteine hydrochloride, a reducing agent, the media
becomes more anaerobic - thus helping to make the environment more conducive to
this organism's growth requirements. YN17, without antibiotics or indicator,
and TPY (recommended on Page 1423, Bergeys Manual, gram-positive organisms)
were also used as growth media. Bifidobacteria responded to culture transfers
and environmental stress by changing their cell morphology and biochemical
reactions.
Method of Analvsis
The sample, either pure or from sewage, was diluted and tested by the MF
procedure (i.e. sewage) or by spread plating (i.e. animal sources) .
The plates were incubated anaerobically for 48 hours at 37° c using BBL
anaerobic Gas Pack anaerobic systems (70304) and indicator strips (70504) in an
anaerobic jar.
Dark blue (YN17) colonies and circular, mucoid, white translucent colonies
(TPY, MRS, 'mil) were ahosen for further examination. .\11 L-if creation
- 296 -
concerning growth, source, etc. was recorded. The colonies were labelled and
inoculated into 5 ml MPS broth with cysteine hydrochloride added just before
use. These tubes were incubated for 48 hours at 37° C anaerobically. From the
tubes, isolated colonies were streaked on different media. The plates were
incubated anaerobiccLLly for 2 days. Each isolate was streaked out on plates
once more to ensure a pure culture. Ihe culture was tested with Gram reagent
(3% potassium hydroxide) catalase reagent (3% hydrogen peroxide) and a slide
was made of the organism using Crystal Violet. (Bifidobacterium is gram-
positive; the stain was used to observe cell appearance) .
The following tests were applied for Bifidobacterium characterization:
Gram stain, catalase, gelatin, bile esculin, Kligler's iron agar, sorbitol,
xylose, cellctoiose, melezitose, arabinose, lactose, mannitol, melibiose,
ribose, mannose, trehcLLose, raffinose, fructose and litmus milk (see Appendix) .
All inoculated tests were incubated at 37*^ C in an anaerobic jar. The sugars
were examined after 24 and 48 hours. A negative control for all the sugar
reactions was incubated along with the other tests. The sugar tubes were
discarded atfter 2 days. Other results were conclusive within 5-7 days. The
appended flow chart was devised by Xu Yan, Arif Somani, R. Cesjardins to help
in the analysis of the results. Please note that numbers on the flow chart
correspond to Table 15.51 (Page 1428) Bergeys Manual, gram-positive organisms.
Table 5 in the paper by Mitsuoka (1982) is also appended as a useful
classification reference.
- 297 -
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Xylose
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Sucrose
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Melibiose
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Dextrin
Starch
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Intiiin
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- 299 -
APPEldX - H
FECKL SlEEETDGDOCr
The following identification scheme (ASM, 1985) has been prepared by Gar/
Horsnell, Ministry of the Environment (1987) and has been employed at the
Ministry of the Environment for fecal streptococcal identification -
Introduction
Ihe fecal streptococci of interest in this study may be illustrated by the
tables given belcw (Modified by Hartman et al. 1966) .
Pecal SLxt^jLocooci
Enterococci
S. faecalis var faecalis
S. faeccLLis var liquefaciens
S. faecalis var zymogenes
S . f aecium
S. f aecium var casseliflavus
S. durans
S. bcvis
Viridans
S. equinus
group
S. mitis
S. scdivarius
Group D
S. avium {GBP D, Q)
MRdia
The fecal streptococcus isolates were picked from recovery media such as m-E
agar and m-Enterococcus agar. (Details of preparations can be found in the
Appendix under Media.) Colonies of all sizes that appeared pink with a blue
halo on m-E and pink to maroon on m-Enterococcus were chosen. Source,
- 300 -
cn 01 < « cn < < _< w
i i i M I 1 2. i
^ n
^ o: z. z
nam
I I I I
5
I
5"
2
>- + +•
S
- 301 -
dilution, media were recorded as well. Care was taken to pidc (with a sterile
loop or needle) only one colony for each isolate sainple.
Method of Collecting Isolates
The colony was streaked onto Brain Heart Infusion (Difco) agar slants and
incubated for 18-24 hours at 37*^ C- The following day, the slants were covered
with sterilized paraffin oil for storage. The isolate would no longer grew but
would be kept free from contamination.
Method of Identif icaticn
Prior to inoculation, the EHI plates were dried under a laminar flow hood
for approximately 15 minutes. The plates, labelled according to the isolate
number, were streaked in 4 quadrants to obtain isolated colonies and incubated
at Zl° C for 18-24 hours. The plates were then examined to determine how many
different types of colonies were present by looking for a) colour; b) colonial
morphology; and c) contamination. Each different type of colony, except the
latter, was transferred onto a EKE plate divided into 6 sections to make a
reservoir (i.e. a spread or patch of growth) . This was done to obtain a larger
volume of bacterial growth for further testing.
The next day, wet mounts of each sample were made and examined under the
microscope. Streptococci are cocci, ovoid to spherical in shape. They occur
in short chains (3-6 cells) on agar or longer chains (more than 6 cells) in
broth or in pairs or individually. They do not appear as tetrads. Next, the
following tests were performed to separate them from other gram-positive cocci. •
Each isolate was tested with 3% H2O2 (hydrogen peroxide) for the catalase
reaction. The test solution was first tested with known stock culture controls
of S . f aecium and Pseudomonas aeruginosa to make sure the solution would
- 302 -
perforTn correctly. Both the catalase reagent and Gram reagent (KDH) were kept
for 2 weeks. S . f aecium is catalase negative; i.e. no reaction when the
culture is tested with reagent while P. aeruginosa is catalase positive.
Introduction of culture to reagent produces biihhles.
For the Gram test, potassium hydroxide (KDH) was used. The culture was
mixed with a drop of NDH for about one minute with a loop. A gram-negative
such as P. aeruginosa breaks down the cell wcLLl and the contents disperse. The
mixture becomes thicker, syrupy and stringy when you raise the loop from the
slide. A gram-positive culture (i.e. Streptococcus) will give no reaction with
KDH.
The isolate was tested on rabbit blood agar (see Appendix) with a single
horizontal streak. The plates were incubated in an anaerobic jar using BBL
anaerobic gas pack anaerobic systems 70304 and indicator strips 70504 for 43
hours at 37° C (To ensure nedia efficiency, test with a known positive (i.e. S.
faecal is v. zvmogenes) and negative (S. f aecium) . A positive hemolysis will
give a clearing of the media around the streaked zone.
All results were recorded on bench sheets. Another isolate reservoir was
streaked onto BHI. Much care was taken in the streaking process since blocd
agar and EHI can be easily contaminated.
Streptococci can be divided into four general groups:
a) the viridans group
b) the lactic group
c) the pyogenic group
d) the fecal or enteric group
There are 6 tests used in this method to help to separate the fecal group
from the others. These are:
1. Bile esculin
2. growth at 45° C - using Todd Hewitt broth (see Media section in
Appendix)
3. growth in 6.5% NaCl
4. arginine
- 303 -
5 . haemolysis
6. antigen serotyping (Group D)
To identify an isolate as an enterococcal group D fecal streptococcus only
the first four tests plus presence of the Group D antigen are required.
The isolates were also inoculated into pyruvate, mannitol, sortose, lactose,
melibiose, gelatin, litmus milk, arabinose and starch agar.
The following observations and conclusions can be made fron the previous
table:
a) S . f aecium variants are usually cirabincse+-, pyruvate- and never gelatirn- or
haeinolytic+. However S. f aecium var casseliflavus which is the only yellow
pigmented organism in the enterococcal group can be arginine^-/-, pyruvate+/-
, (and arabinose^-) .
b) S . f aecalis variants are usually always pyruvate^-, arabinose- and may or may
not be gelatirH", haemolytic+.
It is pcssible for an organism to lose its ability to produce a positive
result (i.e. to ferment a sugar) . Hcwever, an organism cannot gain an ability
to react if it naturally did not have it before, unless the cell obtained a
plasmid-carrying gene through interaction with other, similar organisms.
- 304 -
BinrfipnnfT^I ttp^i- Results
Test
Positive Reacticn
Exanple/Notes
bile
arginine with paraffin oil
(anaerobic)
6.5% NaCl grcwth
arabinose fermentation
lactose fermentation
pyruvate f ennentation
mannitol fermentation
sorbose
litmus miUc
gelatin
Todd Hewitt
melibiose
blood
starch agar
blackening
pink
(all Group D are
positive) except
S. avium and sometimes
S. faecium var
casseliflavios
grcwth
yellow
S. faecium
yellcw
yellcw
S. faecalis variants
yellow
S. avium
ciad, alkali
curds/reduction
liquefaction
S. faecalis liquefaciens
S. faecalis zymogenes
grcwth
yellcw
S . faecium
clearing
S . faecal is
zymogenes
var
S. bovis
Serology
By looking at the profile of all the results it was possible to make an
identification for each isolate. If, by chance, the first 5 tests produced a
negative result, it was necessciry to perform a serology test to see if Group D
was present.
- 305 -
Group-specific antigens are usually carixihydrate structural ccitponents of
the cell wall. Iliese antigens can be extracted in soluble form and identified
by precipitation with hamologous antisera.
There are a number of ways to extract these antigens from the cell wcLLl
including:
1. Hot HCl extraction
2. Hot fonnamide extraction
3. ■ Autoclave extraction
4. Sonication, and
5. Enzyme extraction
Each extraction method has certain advantages and disadvantages.
Generally the autoclave extraction method and the enzyme extraction method are
simple yet reliable procedures for all groups including Group D. Some Group D
streptococcus species (i.e. S. bovis. S. eouinus and S . avium) , however,
contain relatively snail amounts of this antigen and these may require more
severe extraction procedures (i.e. sonication) .
The antigen-antisera precipitation reactions can be performed in various
ways including:
1. Capillary precipitin test
2. Slide agglutination reaction
3. Electrophoretic methods.
Probably the simplest method to employ is the slide agglutination
procedure whereby group-specific antibody coated latex particles are reacted
with the antigen extract.
There are commercially prepared kits available which provide the enzyme
for an enzyme extraction, a reaction slide and antibody coated latex particles
for various serogroups (generally groups A, B, C, D, F, & G) . These latex
particles can ailso be reacted with extract frcrn any other extraction procedure.
- 306 -
The methcxi is as follows:
1. A pure isolate is inoculated into EHI broth and incubated for 18-24 hours
at 37° C. ("Hie broth is brought up to 1% glucose by adding 3g/l and put
in suitable screw cap tubes for centrifugation. )
2. Next day, the broth, culture is centrifuge! at 3000 rpra for 10 minutes to
pack the cells.
3. Hie clear liquid broth (supernatant) may be reacted with the group D
antisera (Group D antibody coated latex particle suspension) (Streptex
Latex Group D suspension Wellcome Diagnostics) to determine the presence
of the Group D antigen. A drop of the suspension is placed on a tile to
which a drop of supernatant is added. "Hie tile is placed on a Variable
Speed Rotator. Observation is made. If agglutination occurs, the
reaction is recorded as a Group D positive. If it doesn't take place, the
extraction procedure continues.
4. The supernatant is carefully decanted from the tube and put in
disinfectant.
5. Five 5inL of physiological sciline (0.35% NaCl) is added to the tube to
resuspend the cells.
6. The tube is once again centrifuged at 3000 rpm for 10 minutes to pack the
cells.
7. The clear saline is decanted into a disinfectant (Dettol) .
8. Then 0.4 ml extraction enzyme (Streptex Extraction Enzyme S . griseus
Wellcome Diag. ) is added to the tube which is shaken to resuspend the
cells.
9. The tubes are incubated at 37° C (water bath) for 1 hour.
10. Following this, the tubes are autoclaved for 30 minutes 15 psi (121° C) .
11. The tubes were cooled and centrifuged at 3000 rpm for 10 minutes.
12. The clear liquid phase is tested for the presence of the group D antigen.
1 drop clear extraction liquid is added to 1 drop group D latex
suspension. A group D positive reaction is indicated by agglutination
(granulcir appearance in mixture) . If the mixture remains clear, the
isolate is group D negative.
- 307 -
AtVtMlLX - I
PSEDEOCHaS AEPDGINCSA.
Introducticri
Pseudcmonas aeruginosa can be found in soil, water, sewage, plants and the
mammalian gut. The organism is pathogenic for hxjmans, plants, insects and some
animals. Since nutritioncLL requirements are simple, and the organism can majce
use of organic ccnpounds, and exist in water of ambient temperature,
Pseudomonas aeruginosa is commonly found in hospitcLL wards. Pseudomonas
aeruginosa produces many toxins and enzymes.
Since Pseudomonas aeruginosa is an opportunistic pathogen, and has been
the cause of severail infections carried through contaminated waters to a
vulnerable host. Pseudomonas aeruginosa has been shown to be the cause of
outer ear infections in swimmers (Seyfried and Fraser, 1978) .
A count of 10 organisms/ 100 ml indicates recent fecal pollution (I^E,
1983) .
The medium used for Pseudomonas aeruginosa enumeration is m-PA (Levin and
Cabelli) (Appendix) . The antibiotics ;:sed in the medium and the high
incubation temperature, help make the medium selective for Pseudomonas by
inhibiting growth of other heterotrophic bacteria.
Method of Sanpling
The sample was tested using the membrane- filtration technique and
dilutions were made as required.
The m-PA plates were incubated for 48 hours at 41.5 + 0.5° C. The plates
were placed, inverted, in a cakette moistened with paper toweling.
- 308 -
Pseudomonas colonies appeared flat, spreading, brownish-green, or tan.
Typical colonies were confirmed by streaking them on Skim Milk Agar (Appendix)
with incubation at 37° C for 48 hours. A positive result was indicated by a
clearing of the n^yjiq and a fluorescent pigment. "Hie culture was then
transferred onto a BHl slant and numbered.
Analysis of PsaJdancnas Isolates
The isolates were streaked fran the BHl slants onto nutrient agar (Difco)
to obtain isolated colonies. These plates were incubated 18-24 hours at 37° C.
The next day, the plates were examined to determine whether the plate had only
one type of colony. If the organisms on the plate appeared as a pure culture,
the colonies were tested with:
a) Gram reagent which was tested against a known positive and negative
control. For example. Streptococcus ffaecium) (G+) was used as a negative
(not sticky) and our Pseudomonas aeruginosa (G-) control was used as a
positive (sticky, syrupy) .
b) Oxidase reagent. On a filter paper (Whatman #1) which was cut to fit
inside a petri dish, the oxidase reagent was applied to the paper and
allowed to dry. (If the oxidase appeared blue from the storage bottle, it
was discarded) . A colony was taken from the plate with a wooden
applicator and stamped onto the paper. A positive reaction resulted when
the spot turned blue, violet. This occurred a few minutes after being
tested. Results were recorded. In addition, the culture was also
streaJced on acetamide slants and skim milk agar.
The acetamide tubes were incubated at 37° C for 72 hours. A positive
appeared as bright pink.
The skim milk agar comprised 4 different tests:
a) A cleeiring of the medium surrounding the streaked zones (+) ;
b) The culture appeared to fluoresce under an Ultra Violet light source
(short wavelength) . These stains produced water-soluble pigments
(pyoverdins) . Pyoverdin production is dependent on nutritional factors.
- 309 -
c) The growth on the plate appeared as yellow-green to brown (pyocyanin
pigment production) +.
The pyocyanin production, which is a blue water-soluble non- fluorescent
pigment is excreted by Pseudoncnas aeruginosa into the medium.
The greenish appearance is caused fay the presence of the (blue) pyocyanin
and the yellcw pyoverdins.
d) Pseudomonas aeruginosa strains possess a grape-like odour.
Sanple Prepeiraticn for Serotyping
1. All cultures showing positive restiLts were sub-cultured to reservoirs on
Nutrient agcir (Difco) with a sterilized swab.
Incubation of the inverted plates was for 18-24 hours at 37<^ C.
2. From the reservoirs, thick suspensions of growth were made into 10 mL of
sterile 0.85% NaCl (Appendix) . The tubes were autoclaved for 30 minutes
at 121° C with a slow exhaust.
3. The autoclaved suspension was centrifuged at 1000-2000 rpn for 10 minutes
and the supernatant was discarded.
4. The pellet was resiispended in 0.75 mL of a solution consisting of 0.85%
NaCl and 1/10,000 merthiolate (Appendix).
Dilution of Sera
The following steps were followed using the 17 sera from the Pseudomonas
aeruginosa antisera kit (Difco) :
1. Resuspended sera by adding 1 mL sterile distilled water to dissolve
contents.
2. Sterilize approximately 20 screw cap tubes.
3. Pipet 0.9 mL of the 0.35% NaCl -merthiolate solution into each tube.
(label tubes 1-17) .
- 310 -
4. Using a clean pipet each time, pipet 0.1 mL of sera into the appropriate
tube.
Testing the Sera
Every day, before the samples were tested the following procedure was followed.
(Both positive and negative controls were tested first) .
1. Antiserums 1-17, as already described, were diluted 1:10 (0.1 mL antiser:a
-I- 0.9 mL NaCl and merthiolate solution) .
2. Antigens 1-17, which were already in solution were mixed (0.1 mL antigen
and 0.9 mL rabbit sera*. *Ratbit serum was first diluted 1/10) .
Positive Oontral
1. Place a drop of antiserum 1 on tile.
2. Place a drop of antigen 1 on top.
3. Next, put the tile on a Variable Speed Rotator (Yankee 070504, Clay Adams,
Parsippany, MJ) so that it will rotate gently. Leave for a few minutes.
There should be agglutination. If however, there isn't, use the antigen
full strength (not diluted with rabbit serum) . Lack of agglutination can
occur if there is too much organism present. A prozone is created
(meaning an excess of antigen) , thus no agglutination occurs.
4. Each antisera should be tested with its corresponding antigen.
Testing Collected Isolates
1. Place a drop of each antisera onto a separate square of the tile.
2. Add the diluted, prepared and mixed sample - one drop per square.
3. Record tile and sample number for referral.
4. Place on the Rotator.
5. Observe closely after a few minutes for agglutination (forming of
granules) .
6. Record results.
- 311 -
Negative CJcntrol
1. A drcp of Antiserum 1 is mixed with a drop of antigen 2.
Result
Antigen 1 + Antiserum 1 +
Antigen 2 + Antiserum 1
Antigen 2 + Antiserum 2
Antigen 1 + Antiserum 2
Antigen 3 + Antiserum 3 +
Antigen 1 + Antiserum 3
(or any except 3)
It is important that no agglutination occurs among heterologous mixtures.
Antisera can be kept in t±ie refrigerator (when not in use) for 5 days.
Antigens made with rabbit sera should be prepared fresh daily.
Iferthiolate (Thimerosol) is a preservative and antiseptic used to prevent
contamination of the antisera.
All antigens and antisera are tested in this manner.
Precauticns
1. The diluted sera should not be kept longer than 5 days.
2. The 0.35 NaCl + merthiolate solution shouldn't be kept longer than 5-7
days unless it is frozen.
3. Unused NaCl and merthiolate solution may be frozen. All unused reagents
should be refrigerated.
4. Note which sera gives weak reaction with antigen control; i.e. 2,3 give
poor reactions when testing with these sera.
. Make sure you observe the sera mixture quite closely from the time you
place the drops on the tile for at least one minute.
5. Keep tile gently swirling at all times.
- 312 -
APPENDIX - J
MEDIA EraPARATICN
Acetamide
Arabinose (see CS method)
Arginine
Bile esculin
(Rabbit) blood agar
1% Carbohydrate solutions (CS)
Catalase reagent
Crystal Violet Reagent
Gelatin
Grains Iodine
Gram Reagent
Lactose (see CS. method)
Litmus milk
Magnesium chloride
Mannitol (see CS. method)
mr-CP2
m-E
Melibiose (see CS. method)
m-Enterococcus
m-PA
MPS agar
m-Tec
0.85 NaCl and merthiolata solution
Oxidase reagent
Phosphate buffer, stock solution
- 313 -
Riosphate buffer, dilution blanks
Pyruvate broth
Saline
Skim milk agar
6.5% Sodium chloride
SortxDse (see C.S. method)
Starch agar plates
Todd Hewitt broth
TPY
Urease reagent
YN17
Medium - Acetamide Agar Slants
Ingredients:
NaCl 5 . Og
Dipotassium hydrogen phosphate (K2HPO4) 1.4g
Acetamide 10 . Og
KH2PO4 0 . 7g
Mg SO4.7H2O l.Og
Hrenol Red 0.012g
Difco Bacto-Agar* 15. Og
Distilled water lOOOmL
Preparation:
- * Add all ingredients except agar
Adjust pH to 6.8; then add agar
- Heat to 92° C
Autoclave media, empty screw cap tubes, screw caps and syringe at 121° C
for 15 minutes
- Before dispensing under laminar flow, adjust pH to 6.8 i_ 0.2
Slant tubes to dry
Reading:
- The acetamide test is used for Pseudcmcnas aerugincsa identification.
The slant is streaked along the surface and incubated for 3 days at. 35°
C. A bright pink is indicative of a positive reaction
- 314 -
Medium - Ar^ginine Dihydrolase Medium Clhomley)
Ingredients:
Bacto Peptone (Difco) 1-Og
NaCl 5-Og
K2HPO4 0-^g
LrArginine Hydrochloride 10. Og
Ehenol Red 0.01 g
Bacto Agar (Difco) 3.0g
Distilled Water lOOOmL
Preparation:
- Mix ingredients into Distilled water
- Adjust pH to 6.8 if necessary
- Heat to 90°C, to dissolve
- Dispense into tubes (5ml/tube)
- Autoclave 15 min. , 15 psi (121° C)
Inoculation:
- Using a needle pick up growth from plate and stab straight down through
centre of arginine medium. Layer top of Arginine medium with
approximately 1 cm sterile paraffin oil.
Incubation:
- 35° C, up to 5 days
Reading:
- positive, medium changes from orangey-pink to bright pink at any tinie up
to 5 days
- negative, no change
Medium - Bile Esculin Agar
Ingredients:
(Difco) Peptone 5.0g
(Difco) Beef extract 3.0g
(Difco) Oxgall 40. Og
(Sigma) Esculin l.Og
Ferric citrate 0.5g
(Difco) Agar 15. Og
Distilled water 1000 mL
- 315 -
Preparation:
- Mix ingredients into Distilled water
- Heat to 90° C, to dissolve
- Dispense into tubes with screw caps (5 ml/tube)
- Autoclave 15 min. , 15 psi (121° C)
- Cool tubes in slanted position
Inoculation:
- Transfer growth from plate and smear, with loop, over slant
Incubation:
- 35° C, up to 72 hr. (3 days)
Reading:
- Positive, blac3csning of one-half or more of slant at any time up to 72
hr.
- Negative, blackening of less than one-half or no blackening of medium at
72 hr.
Medium - Blood Agar (Rabbit) (5%)
Ingredients:
(Difco) Blood Agar base (powder) (BAB) 40. Og
Rabbit Blood (Defibrinatad) 50 mL
Distilled Water 1000 mL
Preparation:
- Mix BAB powder into Distilled Water
- Heat to 90° C, to dissolve
- Autoclave 15 min. 15 psi (121° C)
- Cool to approx. 50-55° C
- Aseptically add 50 mL defibrinatad rabbit blood
- Pour plates under laminar flow hood
Inoculation:
- 1. Pour plate + aerobic incubation
2. Streak plate + anaerobic incubation
Incubation:
- Suggest, 35° C streak, anaerobic, 48 hours
- 316 -
Reading:
- Three Haeinolytic reactions possible
1. Beta (B) - Complete clearing around colony
2. Alpha (a) - Incomplete clearing around colony with red cells left
intact close to colony
3. Gamma (t) - No Haemolysis
Rai±iit blood is used for hemolysis testing because, even though Streptococci
can produce a positive result on other animal bloods, rabbit blood helps to
differentiate between the fecal streptococci since only S. Faecalis z'/r^ccer.es
produces B haemolysis.
Medium - 1% Cartxshydrate Solution
Ingredients:
(Difco) Heart Infusion Broth (powder) 25. Og
Cartx±iydrate Sol^ (10% Aqueous) 100 mL
Distilled Water 900 mL
Brom Cresol Purple Solution (BCP) 1.0 mL
Preparation:
1. Stock indicator Solution
1.6g BCP in 100 mL 95% EIOH
2. Carbohydrate Solution
Carbohydrate 10. Og
Distilled Water 100 mL
- Mix HIB powder into 900 mL Distilled Water
- Add Carbohydrate Sol^ (100 mL)
- Add BCP indicator (1 mL)
- Dispense into tubes (5 mL/tube)
- Autoclave 10 min, 15 psi (121° C)
Inoculation:
- Using a needle inoculate broth tube
Incubation:
- 35° C, up 72 hr. (3 days)
Reading:
- Positive, colour change from purple to yellow
- Negative, no change
The above recipe can be followed to make the following sugar solutions:
lactose , mannitol .
- 317 -
Sugars such as arabinose, sorbose, melibiose are made slightly differently.
Make the HIB solution with indicator.
Autoclave it with test tubes and caps (separately) and syringe. Filter
sterilize the carbohydrate solution (2) into the HIB after it has cooled.
Syringe the solution into the tubes under the laminar flow.
The media is tested with a positive and negative to check it is working
properly.
The tubes are inoculated with some culture and checked every day for 5 days for
positive results and recorded. After incubation time, the result is recorded
as negative if media remains purple.
Medium - Catalase Solution 3% Reagent
Ingredients:
H2O2 (hydrogen peroxide) 30% ' 5 mL
Distilled Water 45 mL
Preparation:
Keep refrigerated for maximum 2 weeks. Check before use, to make sure it
works properly.
Reading:
- [Jrops of this reagent are placed on a microscopic slide. The solution is
firstly tested with known cultures which would produce positive (bubbles) and
negative (no bubbles) results when the culture is added (no mixing necessary)
to the reagent.
Medium - Crystal Violet Reagent
Ingredients:
A Crystal violet 2.0g
Ethanol, 95% 20 mL
B Ammonium Oxalate 0.8g
in 80 mL distilled water
Preparation:
- Add A to B
- Filter
- 313 -
Medium - Gelatin (12%) •
Ingredients:
(Difco) Heaort Infusion Broth (powder) 25. Og
(Difco) Gelatin 120. Og
Distilled Water 1000 mL
Preparation:
Mix ingredients into distilled water
NB - Add Gelatin very slowly so as not to cause clunping
Heat to dissolve (90° C)
- Dispense into tubes (5 mL/tube)
Autoclave 15 min, 15 psi (121° C)
Inoculation:
- Using a needle stab inoculate through Medium
Incubation:
- 35° C, for 5 days
Reading:
- After incubation remove all tubes to refrigerator (-10° C) , cool until
uninoculated control tube is solid when inverted. (15 min. in
refrigerator)
- Positive, medium remains liquid when inverted after cooling
- Negative, medium remciins solid when inverted after cooling
Medium - Gram's Iodine
Ingredients:
Iodine Crystals l-Og
Potassium Iodide 2.0g
Distilled Water 300 mL
Preparation:
- Dissolve chemicals in a small amount of distilled water. Make up to 300
mL.
Medium - Gram Reagent, 3% KDH
Ingredients:
Potassium Hydroxide l-5g
Distilled Water 50 mL
- 319 -
Application:
- TJiis reagent should be kept maximum 2 weeks refrigerated and al'.v-ays
tested prior to use.
- The reagent should first be tested with (2) controls which would give a
pcsitive and negative result to make sure it is working properly.
- Drops of the reagent are placed on a microscope slide.
- A loop of culture is mivpri with the reagent for approximately 60 seconds.
Reading:
- A Gram positive result (G+) occurs when your mixture does not thicken or
become stringy when you lift the loop from the mixture.
- A Gram negative culture when mixed with WDH becomes syrupy or mucoid.
When the loop is raised frcm the mixture it adheres and becomes stringy.
Medium - Litmus Milk
Ingredients:
(Difco) Litmus Milk Powder 100. Og
Distilled Water 1000 mL
Preparation:
- Weigh litnus milk powder and add it to the distilled water. P-it it on
hot plate just to dissolve.
- Dispense 5 mL per test tube.
- Autoclave for 15 min, 121'C.
The litinus milk test was performed on all fecal streptococci isolates. The
culture was inoculated into the tube and incubated at 37° C and checked for up
to 7 days.
There are many reactions which can occur in litmus milk (which is a mauve,
when uninoculatad)
Reduction - The solution or a part of such will turn straw-like (beige)
Acid Curd - Reduction usually occurs in acid curds. Plus there is a
solid curd, pellet on the bottom of the tube whiah does not
move. There, also, may be a clear whey-like liquid en the
top.
Alkaline Curd - Reduction usually occurs in adkali curds as well. There is
a much softer, pliable curd on the bottom '..*u.ch can move or
run. And on the top, there is a red to maroon liquid,
opaque phase.
Proteinization - Sometimes, what was once an alkali curd can become all
liquid (may be same colour and texture as the top phase m
an alkali curd) .
Medium - Stock Magnesium Chloride Solution
- 320 -
Ingredients:
^SgCl2 38. Og
Distilled Water 1000 mL
Preparation:
- Dissolve MgCl2 in the distilled water.
- Keep refrigerated.
This solution is used to make buffered dilution blanks (along with using stock
Phosphate Buffer solution) .
Use 1.25 mL stock Phosphate Buffer and 5 mL Stock iMagnesium chloride solution
per litre of distilled water.
Dispense into bottles and autoclave 15 min, 15 lb.
Medium - m-CP2
Ingredients:
(Difco) SFP Agar Base 52. 5g
L-cysteine Hydrochloride 0.5g
Distilled Water 1000 mL
Preparation:
- Dissolve at boiling temperature
- Autoclave at 121° C for 15 min.
- Cool to 50° C and then filter sterilize (separately) :
sodium resazurin 2mg
Neomycin 0 . 15g
Sodium Azide 0.2g
indoxyl-B-D-glucoside 0 . 5g
Reading:
- Target colonies (i.e. Clostridium perfringens) are yellow with a black
centre which can extend to circumference and are not surrounded by a blue
halo.
Medium - (IXifour's Modified) m-E
Ingredients:
Peptone ICg
Yeast Extract 30g
Sodium Chloride 15g
Sodium Azide 0.15g
Actidione (= cycloheximide store at 4° C) O.OSg
Agar, Bacto (Difco) 15g
Distilled Water 1000 mL
- 321 -
Preparation:
Heat to 90° C.
Autoclave for 15 min. , 121° C.
After avitoclaving, cool the media to 55-60° C
Add ascepticallv. Add all separately:
for 1000 mL
Nalidixic Acid 240 mg in 3 ml sterile distilled '^rater and 0.2
ml ION NaOH, mix and add to medium
Indoxyl-B-D-glucoside 500 mg in 5 ml ethanol (95%). Mix well; then
add 5 ml distilled water and add to medium
Triphenyltetrazoliura
chloride (TIC) 20 mg add jxjst before pouring.
Add very last '
Final pH 7.1 + 0.1 (adjust to 7.1, if necessary)
Pour media under a laminar flow into square petri dishes
This media is used to enumerate and isolate fecal streptococci.
Membrane filtration is performed on a sample. The filters are placed on the
media (which, because of its very high salt concentration makes it very harsh
on the colonies) . The plates are incubated inverted at 41.5'° C for 48 hr. in
a cakette, moistened with paper towelling.
Target colonies appesir as a pink button surrounded by a blue hcilo.
Medium - m-Enterococcus Agar
Ingredients:
(Difco) Bacto m-Enterococcus Agcir 42. Og
Distilled Water 1000 mL
Preparation:
- *Sterilize spatulas and distilled water with magnetic stirrer for 15 min.
at 121° C
- Allow to cool
- Weigh out m-Enterococcus agar. Flame beaker before adding media to water
- Sterilize a thermometer by immersing it in ethanol and wiping it dry.
Place inside media
- Heat media to 90° C
- Once media reaches 90° C, change ever to another (cold) hot plate. Quick
cool the media by placing it in a cakette with ice. Keep changing the
ice to bring the temperature down rapidly.
- 322 -
- When tlie media has reached 55-60° C, check the pH, adjust (if necessarv')
to 7.2 + 0.2. Pour the media under a laminar flow hood into square petri
dishes.
This media is used to enumerate and isolate fecal streptococci. The media is
placed inverted in a cakette which has been moistened with paper towelling.
Plates were incuisated at 37'° C for 48 hours. Target colonies may be pink to
maroon, and of any size. The plates with approximately 8-150 colonies were
counted. With the presence of 2,3,5 - Triphenyl-Tetrazolium chloride (TTC) all
streptococci appear as pink, maroon, along with background or undesired
colonies.
without using TTC strep, faecium and strep, faecal is variants all appear pink,
maroon. However strep, faecium casseliflavus appears yellow.
Medium - MOE Formula mPA Agar (Sterile Technique)
Use sterile glassware
L-Lysine Monohydrochloride
Yeast Extract
Xylose
Sodium Thiosulphate
Magnesium Sulpiiate, Anhydrous
Sucrose
lactose
Sodium QiLoride
Ferric Ammonium Citrate (green)
Sodium Desoxycholate
Kienol Red
Distilled Water, Sterilized
(Difco) *Agar Bacto
*Adjust pH to 7.60. Then add agar.
Preparation:
- Heat to 93° C until boiling starts to dissolve agar.
- DO NOT AUTOCLAVE
- Cool to 60° C. Add antibiotics and mix thoroughly
- Antibiotics are dissolved in 200 mL sterile distilled water
Antibiotics Amount per litre
5.0g
2.0g
. 2.5g
5.0g
1.5g
1.25g
1.25g
5.0g
0.80g
O.lOg
O.OBg
800 mL (to make 1 litre)
15. Og
Sul f apyr idine
Kanaitiycin sulphate
Nalidixic acid
Actidione (Cycloheximide)
0.176g
0.0085g
0.037g
O.lSOg
- Heat antibiotic mixture to approximately 50° C (no higher) to help
dissolve (if necessary) .
- Check surface pH, adjust, if necessary, to 7.1
- Cool to 50° C (no higher than 60° C)
- Pour into petri dishes
- Final pH 7.1 + 0.1
- 323 -
This media is used for enumerating Pseudomonas aerjainosa count.
Moistened paper towelling is added to a cakette, inverted plates are placed
inside and incubated at 41.5° C for 48 hr. Target colonies may appear brovvn,
black. Tan colonies from the filter are streciked on Skim Milk Agar for
Ps. aeruginosa confirmation.
Medium - MRS Agar Plates
Ingredients:
*Cysteine Hydrochloride 0.3g
Gibco MRS Etoth 52. Og
Difco Agar, Bacto 15. Og
Distilled Water 1000 mL
E>reparation:
- Dissolve 52. Og MRS broth into 1000 mL distilled water.
- Add 15 gm agar. Heat to 90° C to dissolve.
- Autoclave for 15 min. at 121° C.
- After autoclaving, cool media to 55-60° C.
*Asceptically, add cysteine hydrochloride.
- Pour media under a laminar flow hood into round petri dishes 100 mm x
ISram.
This media was used to isolate, cultivate Bifidobacteria eit.her by spread
plating or membrane filtration.
Plates were incubated anaembically in an anaerobic jar using BEL GAS Pack
anaerobic systems (70304) with BBL Gas Pack indicator strips (70504) for 24-
48 hr. at 37° C.
Colonies appeared white, round; opaque and mucoid.
Medium - m-TEC Agar (Thermotolerant E^ coli)
Ingredients:
Proteose Peptone No. 3
Yeast Extract
Lactose
Sodium Chloride
Potassium Phosphate, Dibasic K2HPO4
Potassium Phosphate, Monobasic,
Sodium Lauryl Sulphate
Sodium Deoxycholate
Brcmocresol Purple
Bromo Phenol Red
Difco Agar, Bacto
Distilled Water
5.0g
Difco
3.0g
Difco
10. Og
BCH
7.5g
BEH
[PU4
3.3g
Fisher
KH2P04
l.Og
Fi sher
0.2g
BCH
O.lg
BOi
0.08g
Difco
0.08g
BEH
15. Og
Difco
- 324 -
Preparation:
- Mix all ingredients into 1000 mL distilled water.
- Heat to 90° C to dissolve the agar.
- AL±oclave 15 min./lS P.S.I./121° C.
- Allow to cool to 50° C after autoclaving and pour, asceptically into
sterile plates.
- Firal surface pH should equal 7.1 + 0.1.
Because nt-Tec media is the least selective of the ledia used, a negative
control (passing sterile phosphate buffer through unit) was placed on media
before beginning each new saitple. This ensured the unit and buffer were
sterile.
This media is used for enumerating fecal coliforms and E. coli.
Moistened paper towelling is ridded to a cakette, inverted plates are balanced
in the centre and a jar, frozen with 50 mL tap water is placed at either end of
catette. Cakette is incubated at 44.5° C for 23 ± 1 hr.
Yellcw-yellow green colonies aire counted from a filter with 10-150 colonies
present.
Medium - 0.35% NaCl and Merthiolate Solution
Ingredients:
Merthiolate (refrigerated) O.Olgm
NaCl .35gm
Distilled Water 100 mL
Preparation:
- Dissolve merthiolate, NaCl in distilled water.
- Autoclave for 15 min. , 121° C.
- Refrigerate, when cooled.
This solution is used as a diluting reagent for Pseudomonas Antibody Sera.
To dilute sera (i.e. antisera) 0.9 mL of the above solution is mixed with 0.1
mL sera (i.e. antisera) .
To prepare Pseudomonas aeruginosa isolates for serotyping, after a pure culture
is grown in saline overnight, it is autoclaved and centrifuged. The above
solution (0.75 mL) is added to the pellet and mixed. And this solution is used
for tge slide agglutination test.
Medium - Oxidase Reagent
NNN%^ - Tetramethyl - p - Phenylenediamine 0.25g
Ascorbic Acid 0.025g
Sterile Distilled Water
(warm) not higher than 35° C 25 mL
- 325 -
Preparation:
- NEVER STIR - GENTLZ SHAKE (I.E. RDIAIE)
- * Add ascorbic acid to distilled water to give a 0.1% solution.
- Add 0.25g of NNN%-^ - Tetramsthyl - p - Phenylediamine dihydrochloride
- Store in dark, 30 mL eye dropper bottles at 4^ C.
- Reagent should last 7-14 days, but should not be used if it turns blue.
Hie oxidase reagent drops cure applied to a Whatman No. 1 filter paper --vhich has
been cut to fit a petri dish. The oxidase test is performed once the paper has
dried. The reagent is firstly tested with cultures which would produce a
positive result, and a negative result. A wooden applicator is used to pick 'jp
some culture and pressed on the oxidase paper. A positive is indicated if the
spot turns blue (purple) ; while a negative appears as no change.
Medium - Phosphate Buffer Stock Solution
Ingredients:
Potassium dihydrogen phosphate (KH2PO4) 34. Og
Distilled Water volume up to 1 L.
Preparation:
1. Dissolve KH2PO4 in 500 mL distilled water (in 2 L. beaker)
2. Adjust pH to 7.2 + 0.2 with l.Oti NaOH.
3. Pour this amount into 1 L. graduated cylinder and make up to 1 L. volume.
4. Pour the solution back in to the same beaker and autoclave it at 121° C
for 15 min.
After autoclaving, when cooled, pour the sterilized solution in to a sterilized
1 L. volumetric flask.
May be kept at 4° C for no longer than 1 month.
The above stock solution is used to make:
a) Wash waters for membrane filtration (MF)
Use 1.25 mL stock solution per 1 L- distilled water
Autoclave 15 min., 121° C.
Refrigerate.
b) Dilution blanJ<s for samples for MF or spread plating. Use 1.25 mL stock
phosphate buffer solution and 5 mL stock magnesium chloride per L.
distilled water. Fill dilution blanks with approximately 104 mL/bottles
(99 mL + 4 mL for evaporation) . Autoclave for 15 min. at 121° C.
Refrigerate.
- 326 -
Medium - Pyruvate Broth
Ingredients:
Tryptone 10. Og
Yeast Extract 5.0g
K2HPO4 5.0g
NaCl 5.0g
Pyruvic acid, sodium salt 10. Og
Bnanothymol blue (BTB) 0.04g
Distilled Water 1000 mL
Preparation:
- Mix all ingredients except BTB into distilled water.
- * BTB solution
Mix 0.5g bramothymol blue in 5 mL IN NaOH.
Add 0.4 mL of this solution into basal medium.
- Adjust pH to 7.2
- Dispense 5 mL per tube
- Autoclave 15 min. at 121° C
The streptococcus culture is inoculated into pyruvate broth. The test tube is
inoculated for 24 hours (only) at 35° C. A lime green or yellow represents a
positive result.
Medium - Saline (physiological, 0.85%)
Ingredients:
Sodium chloride 8.5g
Distilled Water 1000 mL
Preparation:
- Dissolve scilt in the distilled water.
- If required in test tubes, dispense before autoclaving.
- Autoclave at 121° C, 15 lb.
- Keep refrigerated.
Medium - Skim MiUc Agar
Ingredients:
Difco Slcim Milk Powder lOOg per litre
Difco Bacto-Agar 15. Og
Distilled Water 1000 mL
- 327 -
Preparation:
- Slowly add skim milk powder to 500 mL distilled water and stir without
heat for 30 min.
- In a separate beaker, slowly add the agar to 500 mL distilled water vvtiile
stirring. Heat the agar solution slowly to 90-92° C (10-12 min.)*
- Autoclave the two solutions separately for 12 min. at 121° C. After
autoclaving, cool the 2 solutions to approximately 55' C then [at which
time, add the skim milk solution asceptically to the agar solution. Stir
the mi>:ed solution for an additional 2-3 min. (Temperature should be 50-
52° C) ] .
- Dispense asceptically into sterile plates
[20 mL in round (100 x 13 inm) plates].
- Prepare a small plate for quality control pH reading.
- Adjust to 6.4 + 0.2 at 25° C (surface reading) .
This media is used to determine many characteristics associated with
Pseudomonas aeruginosa . such as pigmentation, caseinase production,
fluorescence and oxidase reaction. Plates are streaked for isolated colonies
and incubated for 48 hr. at 37° C.
Pseudoinonas aeruginosa usually appears yellow-green, lime green, dark green,
brown on Skim milk agar.
A clearing of the media indicates caseinase production. Under ultra-violer
light source, Pseudomonas aeruginosa colonies fluorese. Using the plate,
provided the sarnple appears pure, it can be tested with the oxidase reagent
(Ps. aeruginosa +) (Please see oxidase Reagent for detailed information) .
Medium - 6.5% Sodium chloride
Ingredients:
(Difco) Heart Infusion Broth (powder) 25. Og
NaCL 65 . Og
Distilled Water 1000 mL
Preparation:
- Mix ingredients into Distilled Water
- Dispense into tubes (5mL/tube)
- Autoclave 15 mi, P.S.I. (121° C)
Inoculation:
- Using a needle pick some growth frcm the plate and inoculate lightly into
the broth - no visible turbidity should be detected after inoculation.
Incubation:
- 35° C, up to 72 hr. (3 days)
- 323 -
Reading:
- Positive, growth (visible turbidity) at any time up to 3 days
- Negative, no visible turbidity at 3 days
Madium - Stcirch Agar Plates
Ingredients: . , .,
(Difco) Blood Agar Base powder' 40. Og
Soluble starch 20. Og
Distilled Water 1000 mL
Preparation:
- Slowly, add 20g soluble starch to 1000 mL distilled water.
- Add 40g Blood Agar Base to the mixture. Heat the solution to 90° C to
dissolve.
- Autoclave 15 min. , 121° C.
- Pour in a laminar flow hood into round petri dishes 100 mm x 15 mm.
- Refrigerate plates.
The fecal streptococci isolates were tested for starch hydrolysis by placing a
1 line streak on a 6 sector plate. The plates were incubated at 37° C for 5
days. At which time they were flooded with Grain's Iodine solution and checked
after 30 min. A clearing of the media indicates a positive starch resiilt.
Ifedium - Todd-Hewitt Broth
Ingredients:
(Difco) Todd Hewitt Broth Powder 30. Og
Distilled Water 1000 mL
Preparation:
- Mix powder into distilled water
- Dispense into tubes (5 ml/tube)
- Autoclave, 15 min., 15 psi (121° C) .
Inoculation:
- For testing growth at 10° C and 45° C using a needle, transfer seme
growth from the plate into the broth - No visible turbidity should be
detected after inoculation.
Incubation:
- 10° C up to 5 days
- 45° C
- 329 -
Reading:
- 10° C and 45° C
- Positive, - visible growth in broth
- Negative, - clear non turbid broth
Madium - TFY
Ingredients:
BBL Trypticase 10. Og
BBL Fhytone 5.0g
Glucose 5.0g
Difco Yeast Extract 2.5g
Tween 80 1 aiL
Cysteine hydrochloride 0.5g
K2HPO4 2 . Og
^^2-6H20 " 0,5g
ZnS04 . 7H2O , 0 . 25g
CaCl2 0.15g
FeCl3 trace
Difco Bacto, i^gar 15. Og
Distilled Water 1000 mL
Preparation:
- Final pH is 6.5 after autoclaving at 121° C for 25 min.
- The media was poured under a lamincur flow hood into round petri dishes
100 mm X 15 inm.
This media was used to isolate, cultivate Bifidobacteria either by spread
plating or membrane filtration.
Plates were incubated anaerobically in an anaerobic jar using BBL Gas Pack
anaerobic systems (70304) with BBL Gas Pack indicator strips (70504) for 43 hr.
at 37° C.
Medium - Urease Reagent
Ingredients:
Urea 10 . Og
Phenol Red Solution 0.5 mL
l.Og of phenol red in 10 mL ethanol (0.25 in 2.5 mL)
Distilled Water 500 mL
Preparation:
- Weigh the phenol red in a weigh boat. Add the alcchol and ni:< zc
dissolve the dye.
- Weigh the urea and add it to the distilled water.
- ?dd 0.5 mL of the prepared phenol red solution to t.^.e 'area soluricn.
- Adjust pH to 5.0 + 0.2
- 330 -
- Store in a pyrex container for up to 2 weeks in the refrigerator.
- Check (and adjust) pH before use.
The urease test is perfomved to determine the presence of E. coli.
After the m-Tec (media used to enumerate Tecal coliforms) is counted, the
filter is placed on a pad, contained in a small petri dish, soaked with urease
reagent (but not dripping) . The filter is left for 15 min. and recounted.
Yellow-yellow green colonies are indicative of E. coli.
Medium - YN17(b) (MF media for Bifidobacteria)
Ingredients:
Yeast extract 20. Og
Polypeptone 10 . Og
Lactose • 10 . Og
Casamino acid 8.0g
NaCl 3 . 2g
Bromocresol green 0.30g
Cysteine hydrochloride 0.40g
(Difco) Bacto-agar 15. Og
Distilled Water 1000 mL
Preparation:
- Stir on medium heat until agar dissolves.
- Autoclave for 15 min. at 121° C.
- Adjust pH to 6.9 + 0.1.
- Cool to 60° C. and add:
Ncilidixic acid 0.03g
Kanamycin sulphate O.OSg
Polymixin B sulphate 0.0062g
Target colonies on YN17 (b) appear dark blue and black.
*YN17 growth media
Follow above recipe. However, do not include bronocresol green or antibiotics.
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